Simulated Migration of European Eel (Anguilla anguilla, Linnaeus 1758)

Promotor Prof.Dr.Johan A.J. Verreth Hoogleraar in de Aquacultuur en Visserij Wageningen Universiteit

Co-promotor Dr. Guido E.E.J.M. van den Thillart Universitair Hoofd Docent, Instituut Biologie, Universiteit Leiden

Promotiecommissie Prof. Dr. Ir. M. W. A. Verstegen (Wageningen Universiteit) Dr. A. J. Murk (Wageningen Universiteit) Prof. Dr. S. E. Wendelaar Bonga (Radboud Universiteit Nijmegen) Dr. S. Dufour (National Center of Scientific Research, MNHN Paris, France)

Simulated Migration of European Eel (Anguilla anguilla, Linnaeus 1758)

Vincentius Johannes Theodor van Ginneken

Proefschrift

Ter verkrijging van de graad van Doctor op gezag van de Rector Magnificus van Wageningen Universiteit Prof.Dr. M.J.Kropff in het openbaar te verdedigen op woensdag 14 juni 2006 des namiddags te half twee in de Aula

Van Ginneken, V.J.T. Simulated migration of European eel (Anguilla anguilla, Linnaeus 1758)

PhD Thesis, Wageningen University, The Netherlands With ref.- With summary in English, and Dutch ISBN: 90-8504-456-1

Daarom wordt mij verschillende malen te verstaan gegeven, dat ik, waar ik zo stellig het ontstaan door voortteling beweer, de wijze van voortteling van de alen zou moeten aantonen, hoofdzakelijk daarom, omdat het grootste deel van de mensen stellig gelooft, dat de alen zonder het middel der voortteling voortkomen; alsof ik in staat moest zijn, in geval ik zodanige bovengenoemde stellingen volhield, op te lossen al hetgeen omtrent genoemd onderwerp mij werd voorgelegd. Hoewel het veld van de dingen die tot nog toe in het duister verborgen zijn, zo ruim en wijd is. Niettemin heb ik enige jaren reeds alle moeite gedaan om, indien het mogelijk was, de voortteling der Alen te ontdekken en haar voor de ogen van de Wereld te plaatsen. (Antoni van Leeuwenhoek, Brief No. 115, 18 september 1691).

Contents

Samenvatting 1

Summary 7

Chapter 1. General Introduction: The European eel (Anguilla Anguilla L.) its 12 lifecycle and reproduction; possible causes for decline of eel populations.

Chapter 1. Aims and outline of the Thesis 32

Chapter 2. Microelectronic detection of activity level and magnetic orientation of 35 yellow European eel, Anguilla anguilla L., in a pond.

Chapter 3. Silvering of European eel (Anguilla anguilla L.): seasonal changes of 50 morphological and metabolic parameters.

Chapter4. Endocrine and metabolic profiles during silvering of the European eel 69 (Anguilla anguilla L.).

Chapter 5. Direct calorimetry of free moving eels with manipulated thyroid status. 85

Chapter 6. Endurance swimming of European eel 97

Chapter 7. Acute stress syndrome of the yellow European eel 109 (Anguilla anguilla Linnaeus) when exposed to a graded swimming-load

Chapter 8. Eel fat stores are enough to reach the Sargasso 125

Chapter 9. Eel migration to the Sargasso: remarkably high swimming efficiency 129 and low energy costs.

Chapter 10. Hematology patterns of migrating European eels 146 and the role of EVEX virus

Chapter 11. Presence of eel viruses in eel species from various geographic regions 160

Chapter 12. Effects of PCBs on the energy cost of migration and blood parameters of 167 European silver eel (Anguilla anguilla, Linnaeus 1758)

Chapter 13. A 5,500-km swim trial stimulates gonad maturation 189 in the European eel (Anguilla anguilla L.)

Chapter 14. Recommendations for protection of the eel populations and 212 suggestions for future research

Annex 1. The European eel (Anguilla anguilla, Linnaeus), its lifecycle, 229 Evolution and reproduction: a literature review

Annex 2. Gonad development and spawning behavior of 279 artificially-matured European eel (Anguilla anguilla L.).

Annex 3. Publicity disseminations (news papers, TV, Radio etc.) 299

Annex 4. List of publications 301

Annex 5. Curriculum Vitae 305

Dankwoord 307

Samenvatting

Samenvatting proefschrift “Gesimuleerde migratie van de Europese aal (Anguilla anguilla L.)”

Introductie Over de laatste 25 jaar is de populatie van de Europese paling is zo'n sterke mate afgenomen dat er grote zorgen zijn ontstaan om zijn voortbestaan op de lange termijn. Populaties van volwassen dieren begonnen af te nemen vanaf 1940 in grote delen van het Europees continent, terwijl het recruitment (aanwas via golven van glasaal) vanaf de jaren tachtig van de vorige eeuw zijn afgenomen. Tot op heden zijn er geen signalen van herstel en dit fenomeen kan gesignaleerd worden over het hele levensgebied van de Europese paling. Een parallelle ontwikkeling kan worden geobserveerd voor de nauw verwante Amerikaanse paling (A.rostrata) en de Japanse paling (A.japonica). De Europese paling (Anguilla anguilla L.) is een katadrome vissoort met zijn paaigebieden duizenden kilometers ver in de oceaan. Een belangrijk aspect van de reproductie van de Europese paling is de enorme afstand die zij moeten zwemmen om hun paaigebieden te bereiken. Na het verlaten van de Europese kusten moeten ze 5000-6000 km zwemmen om de Sargasso Zee te bereiken. Van deze zee wordt aangenomen dat hier de paaigronden liggen. Om deze enorme afstand af te leggen moeten de alen 6 maanden lang bij een 0.5 lichaamslengte per seconde zwemmen wat een indrukwekkende lange termijn zweminspanning vereist. Daarnaast zijn grote energievoorraden gekoppeld met lage energiekosten voor transport vereist. Hieraan kan de hypothese worden toegevoegd dat lange termijn zwemmen een belangrijke voorwaarde kan zijn voor reproductie. In dit proefschrift hebben we de capaciteit van Europese paling onderzocht om over deze lange afstand te migreren. De zoetwaterfase van groei, geslachtsdifferentiatie en ‘schier’ worden, (een preadaptatie aan zijn oceanische fase en terugkeer naar zijn paaigronden) voor de migratie bepaalt uiteindelijk de kwaliteit van de ouderdieren. Deze periode in het zoete water kan een periode van 5-50 jaar beslaan. De kwaliteit van de ‘habitat’ (woonomgeving) en de kwaliteit van habitatfactoren zoals voedseltekort (wat leidt tot verminderde vetvoorraden), virussen en giftige stoffen (zoals PCB’s = polychloorbiphenyls) is belangrijk voor de zwemcapaciteit van de ouderdieren en de kwaliteit van de geslachtsproducten. In dit proefschrift zullen we de factoren die de levenscyclus van de Europese paling beschrijven, dit om meer begrip te krijgen voor de mogelijke factoren die betrokken zijn bij de afname van palingpopulaties en die betrokken zijn bij de reproductie.

De zoetwaterfase, oriëntatie op het aardmagnetisch veld: In de literatuur zijn verschillende publicaties te vinden van veldstudies in bassins telemetrische studies, studies met sterke kunstmatige magnetische velden die de natuurlijke voorkeursrichting van paling overtreffen, die aangeven dat oriëntatie wordt bewerkstelligd door kenmerken van het aardmagnetisch veld. Ook de observatie van magnetische substanties in de schedel en botten van palingen ondersteunt in sterke mate deze zienswijze. Wij bestudeerden de circadiane (24-uurs) en maandelijkse activiteit, het distributiepatroon, en oriëntatie op het aardmagnetisch veld van niet-schiere (niet-migrerende) vrouwelijke paling op een zoetwatervijver door middel van microchips geïmplanteerd in hun spieren. Detectoren voor de microchips werden gemonteerd in buizen en deze worden op de vijver geplaatst om vast te stelen of palingen zich oriënteerden ten opzichte van het aardmagnetisch veld. Gebaseerd op de frequentie van het bezoeken van de buizen (corresponderend met het zoekgedrag naar een schuilplaats), gaven de data aan dat de aanwezigheid van de palingen in

1 Samenvatting

de buizen geleidelijk afnam gedurende de duur van de studie. Daarnaast zagen we meer activiteit gedurende de nacht in de eerste paar maanden. Er was een seizoenscomponent in het oriëntatie mechanisme met een significant lagere voorkeurscomponent in de zomer in vergelijking tot de herfstperiode, de periode waarbij de migratie op gang komt. Een voorkeurspositie voor buizen georiënteerd in de Zuidzuidwestelijk richting (de richting van de Sargasso Zee) in de herfst suggereert een oriëntatie gebaseerd op het aard magnetisch veld.

De zoetwaterfase: het schier worden De overgang van niet-schiere (niet-migrerende) naar schieraal (migrerend) wordt schier worden (‘silvering’) genoemd, en dit proces neemt plaats kort voor migratie. De mechanismen betrokken bij het in gang zetten van het schier worden zijn grotendeels onbekend. Ook een duidelijke beschrijving van de verschillende stadia, die de metamorfose karakteriseren ontbreken. Tot zeer recent werd het proces van schier worden voornamelijk gebaseerd op morfologische kenmerken en er werd een opsplitsing gemaakt in twee gescheiden stadia: ‘schier’ en ‘niet-schier’. Deze classificatie nam geen mogelijk voorbereidingsstadium in ogenschouw. Wij beschrijven hormonale profielen van Europese paling tijdens het proces van ‘schier’- worden. Wij hebben ook gebruik gemaakt in de beschrijving van fysiologische kenmerken als lichaamssamenstelling en bloedsubstraten. Deze transformatie gebeurd in associatie met hoge hormonale concentraties van testosteron (T), oestradiol (E2), cortisol maar niet met hoge concentraties van schildklierhormonen (TH) en groeihormoon (GH) welke een maximale activiteit hebben in het voorjaar en een minimale activiteit in de zomer en de herfst. In tegenstelling hiermee worden in de herfst hoge concentraties van cortisol gevonden welke een grote rol spelen in de mobilisatie van metabole energie van lichaamsvoorraden, naar migratie activiteit en de groei van de gonade. Gebaseerd op een statistische methode genaamd ‘Principal Component Analysis’ met fysiologische morfologische en endocrinologische parameters kan er geconcludeerd worden dat de overgang naar schier worden gradueel is en dat de paling door verschillende stadia gaat.

De zoetwaterfase, de rol van schildklierhormoon Bij amfibieën zoals kikkers wordt de metamorfose van larve naar volwassen dier gereguleerd door schildklierhormoon. Voor andere koudbloedigen zoals vissen, wordt ook een rol voor schildklierhormoon aangenomen zoals bij zalmen gedurende de ‘parr-smolt’ transformatie. In onze studie van de jaarcyclus hebben we echter waargenomen dat de concentratie van schildklierhormonen erg hoog is in het voorjaar maar niet in de herfst tijdens het proces van ‘schier’ worden (silvering). Gebaseerd op dit gegeven kunnen we mogelijk concluderen dat de schildklierhormonen mogelijk niet betrokken zijn bij het schier worden. Een andere mogelijkheid is dat hun actie calorigeen is en betrokken bij de controle van de stofwisselingssnelheid zoals bij vogels en zoogdieren het geval is. Wij hebben met directe calorimetrie de totale warmte productie gemeten in vrij bewegende palingen met verschillende thyroid status met een nauwkeurigheid van 0.1 mW. Hyperthyroidisme werd geïnitieerd door injectie van T3 en T4 hormonen terwijl het effect van hypothyroidisme bestudeerd werd door de dieren bloot te stellen aan phenylthioureum. De resultaten laten voor het eerst op het niveau van het organisme zien, gebruik makend van de techniek van directe calorimetrie, dat nog de totale warmte productie nog de totale zuurstofconsumptie in palingen beïnvloed wordt door hyperthyroidisme. Hieruit kunnen we concluderen dat een stimulerend effect van thyroid hormonen op de thermogene stofwisselingssnelheid niet optreed bij een koudbloedige soort als de Europese paling.

2 Samenvatting

Het nieuwe type Blazka zwemtunnel Wij hebben een Blazka zwemtunnel van 127 liter ontwikkeld met een totale lengte van 2.0 meter en een lengte van het zwemcompartiment van 1.15 meter om de lange duur zwemcapaciteit van schieralen met een lengte van 80-90 centimeter te testen. Wij hebben met een zeer nauwkeurig Laser-Doppler systeem de homogeniteit van de stroming in de zwemtunnels aangetoond. De eigenlijke doorstroming werd gemeten op verschillende dwarsdoorsneden van de tunnel en op verschillende plaatsen van de wand. Een lineaire verband werd gevonden tussen het aantal omwentelingen per minuut van de motor en gemeten water snelheid. Deze lineairiteit bleef bestaan tot 0.9 meter per seconde. De waterdoorstroming vanaf 40 mm van de wand tot het midden van de tunnel bleef binnen een paar procent van de ingestelde waarde. Hieruit kunnen we concluderen dat vissen met een dwarsdoorsnede van > 40 mm niet in de grensvlaklaag kunnen zwemmen. De palingen gebruikt in de verschillende zwemstudies hadden nog meer ruimte nodig vanwege de amplitude van hun staartslag. Daarnaast observeerden wij dat de kop van de palingen tussen de 50 en 100 mm van de wand af bleef.

Migratie Lange termijn zwemexperimenten over 5.500 km met virusnegatieve Europese paling tonen aan dat palingen erg efficiënte zwemmers zijn. Palingen hebben een vetpercentage van 10- 28%, met een gemiddelde van 20% en dit is overduidelijk hun belangrijkste energievoorraad. 40% van de totale vetreserve van schieraal wordt gebruikt om te zwemmen, 60% blijft over voor de aanleg van de gonade. Dieren met een vetpercentage lager dan 13% vet zijn niet in staat om 6000 km te zwemmen. In vergelijking tot andere vissoorten zoals de zalm zijn palingen erg efficiënte zwemmers met energie kosten voor migratie die 4-6 keer lager liggen dan die voor salmoniden. De ‘Kosten voor Transport’ (Cost of Transportation, COT) voor een paling zijn 0.68 kJ.kg-1.km-1 terwijl de COT voor forel 2.73 kJ.kg-1.km-1 is. Het geschatte vetverbruik van een volwassen paling om de Atlantische Oceaan over te steken (6000 km) bedraagt 29% van zijn vetvoorraden. Dit correspondeert met 58 gram vet per kg paling terwijl dit voor zalm 300 gram per kg zou bedragen. Op dit moment wordt niet begrepen waarom palingen zulke efficiënte zwemmers zijn. In toekomstige studies moet de hydrodynamica verklaren hoe ‘undulatory’ zwemmen (karakteristiek voor anguilliform = palingachtige beweging) werkt. Hiervoor moeten twee vragen worden beantwoord: a) het spierontwerp: welke spierrangschikking is het meest geschikt om het lichaam te buigen? b) hoe zet de vis spiervermogen om in zwemvermogen?

Effecten van omgevingsfactoren op de migratie Wereldwijd zijn palingpopulaties in sterke mate afgenomen over de laatste twee decennia van de vorige eeuw. De exacte oorzaak voor dit fenomeen is onbekend maar mogelijke oorzaken zijn: PCB’s, virussen en verminderde vetvoorraden. Om te onderzoeken of deze factoren een effect hebben op de zwemprestatie en -capaciteit van Europese paling werden zwemexperimenten uitgevoerd in 22 grote 127 liter zwemtunnels in het laboratorium.

PCB’s: De resultaten van onze studie gaven 5 belangrijke observaties. Ten eerste verliezen aan PCB blootgestelde dieren minder gewicht en bezitten ze een lagere glucose- en cortisolspiegel (alleen zwemmende dieren) in vergelijking tot de niet blootgestelde Controle dieren. Ten tweede, zijn PCB concentraties op een vetbasis 2.7 keer zo hoog in zwemmende dieren in vergelijking tot rustende dieren. Ten derde heeft PCB-blootstelling het effect dat het zuurstofverbruik van de zwemmende, aan PCB blootgestelde dieren vanaf 400 km (18 dagen) significant verlaagd is en dit effect neemt in de tijd toe. De Kosten van Transport (COT, [mg

3 Samenvatting

-1 -1 O2. kg . km ]) zijn significant lager in PCB-blootgestelde dieren vanaf 100 km tot en met 800 km. Daarnaast was de standaard metabole snelheid (basaalstofwisseling) twee dagen gemeten na de laatste zwemactiviteit significant verlaagd in PCB-blootgestelde dieren. Ten vierde is de milt vergroot in de PCB-blootgestelde zwemdieren maar niet in de PCB- blootgestelde controle dieren. Ten vijfde kunnnen schieralen makkelijk rusten in zeewater en zwemactiviteit volbrengen in zoetwater maar niet bij een combinatie van deze twee stressfactoren. Plasma-pH, ionen niveau’s (natrium en kalium), plasma melkzuur, hemoglobine en hematocriet waren niet beinvloed door PCB- blootstelling. We concluderen dat PCB-blootstelling interfereert met de energiestofwissling van schieralen in zeewater waarbij deze schijnt te interfereren met de cortisolcontrole over de koolhydraat stofwisseling. Dit effect was groter in zwemmende dan in rustende dieren.

Virussen: EVEX (‘Eel-Virus-European-X’), HVA (Herpesvirus anguillae) and EVE (Eel Virus European) werden waargenomen in wilde en gekweekte Europese palingen (Anguilla anguilla) uit Nederland, EVEX en EVE in gekweekte paling uit Italië, en EVEX in wilde paling uit Marokko. EVEX werd ook geïsoleerd uit wilde paling uit Nieuw Zeeland (A.dieffenbachi). Jonge aal (A.anguilla) verzameld uit palingkwekerijen in Nederland was voornamelijk geïnfecteerd met HVA. Wijd verspreide infectie van de palingpopulatie met bijvoorbeeld EVEX virus kan het gevolg zijn van ongelimiteerd palingtransport tussen de verschillende continenten. Daarnaast toonden wij in grote zwemtunnels gedurende een gesimuleerde migratie aan dat paling geïnfecteerd met EVEX bloedingen op het lichaam en bloedarmoede kreeg, en stierf na 1000-1500 km. In tegenstelling tot dit gegeven zwommen virus-negatieve dieren 5.500 km, de geschatte afstand van de Europese kust tot de Sargasso Zee. De virus-positieve dieren vertoonden een daling in hematocriet gerelateerd tot de zwem afstand. De virus-negatieve dieren vertoonden een iets verhoogd hematocrietgehalte. De geobserveerde veranderingen in plasma lactaatdehydrogenase (LDH), Totaal Eiwit en Aspartaat aminotransferase (AST) zijn indicatief voor een ernstige virusinfectie. Het is dus mogelijk dat virusinfecties en verontreiniging met PCB’s kunnen bijdragen aan een afname van de palingpopulaties.

Reproductie Wij observeerden dat in hormoon-behandelde Europese paling afpaaigedrag kon worden geïnduceerd Dit gedrag van de alen was collectief en vond simultaan plaats corresponderend met afpaaigedrag in een groep. Dit is de eerste keer dat afpaaigedrag is geobserveerd en geregistreerd voor palingen en het opent nieuwe perspectieven voor onderzoek in de nabije toekomst. Een ander belangrijk onderzoeksresultaat met betrekking tot de reproductie van deze soort was de observatie dat in drie jaar oude (juveniele) Europese palingen welke 173 dagen zwommen in Blazka zwemtunnels, waarbij ze een afstand van 5500 km overbrugden, aan het eind van de rit de maturatieparameters 11-ketotestosteron, hypofyseniveaus van lutheinizing hormoon (LH) en plasmaniveaus van oestradiol hoger waren in de zwemgroep (zij het niet significant) in vergelijking tot de restgroep. Daarentegen was de eidiameter significant groter in de zwemgroep in vergelijking tot de restgroep. Gebaseerd op deze observaties concluderen wij dat een periode van langdurig zwemmen een fysiologische stimulus kan zijn voor het in gang zetten van de maturatie. Experimenten in toekomstige studies met volwassen virusvrije dieren moeten in de toekomst definitief aangeven of langdurig zwemmen een natuurlijke prikkel is voor de gonadenmaturatie van de Europese paling.

4 Samenvatting

Panmixia, moleculair werk De hypothese dat alle Europese palingen naar de Sargasso Zee zwemmen voor reproductie en bestaan uit één enkele random afpaaiende populatie, de zogenaamde ‘panmixia’ theorie, was tot voor kort wijd geaccepteerd. Echter, gebaseerd op observaties uit het veld, morfologische parameters en moleculaire studies zijn er indicaties dat de claim van de Deense Bioloog Johannes Schmidt uit 1923 van een unieke afpaaiplaats in de Sargasso Zee voor de Europese palingpopulatie een te sterke bewering kan zijn. Recente moleculaire studies aan de Europese paling (een overzicht van het werk van de verschillende auteurs is gegeven in dit overzichtsartikel) zijn indicatief voor een genetisch mozaïek bestaande uit verschillende geïsoleerde groepen. Dit gegeven leidt tot een verwerping van de panmixia theorie. Echter, de laatste uitgebreide genetische studie van onze Belgische collega's van de Universiteit van Leuven laat zien dat de geografische component van de genetische structuur gebrek vertoont aan temporele (in de tijd bezien) stabiliteit hetgeen benadrukt dat er temporele replicatie (herhaling) moet plaatsvinden bij het bestuderen van deze zich ver verspreidende mariene soort. Panmixia kan in de toekomst toch nog tot de mogelijkheden behoren.

Bedreigde diersoort Gedurende de laatste twee decennia van de vorige eeuw zijn palingpopulaties afgenomen met 90-99%, mogelijk ten gevolge van een synergie tussen menselijke activiteiten en oceaan fluctuaties, en deze afname heeft ertoe geleid dat het een bedreigde diersoort is. Drie miljoen jaren slaagde de katadrome Europese paling erin om te overleven en zijn karakteristieke levensstijl te handhaven. Deze levensstijl kan worden gekarakteriseerd door afpaaigebieden in de oceaan, (mogelijk de Sargasso Zee) en zijn juveniele levensfase van groei en sekse differentiatie in het zoete water van het Europese continent. De volgende aanbevelingen kunnen worden gedaan om een totaal uitsterven van deze soort te voorkomen: a) Het verminderen van de visserijdruk door in de binnenlandse wateren natuurreservaten in te stellen om deze territorium-gebonden vissoort te beschermen. b) Het ontwikkelen van vroege waarschuwingssystemen bij waterkrachtcentrales om een groot deel van de migrerende schieraal tijdens het migratieseizoen in de herfst te beschermen. c) PCB verontreiniging moet in alle grote watersystemen bemeten worden en gebieden met lage PCB niveaus moeten worden beschermd. d) Om het verspreiden van parasieten en virussen te voorkomen moeten internationale wereldwijde instructies voor sanitaire standaardisering voor het transport van aquatische organismen geïntroduceerd worden. e) Onderzoek naar kunstmatige voortplanting van de paling moet uitgebreid worden door zich te concentreren op toedieningstechnieken van hormonen. Er moet meer onderzoek worden gedaan naar het natuurlijk afpaaigedrag van paling en de rol van feromonen. En verder moet het onderzoek zich concentreren op het begrijpen van de natuurlijke stimulus voor reproductie. f) Moleculaire technieken om cellen in te bouwen met genen (gentherapie) die maturatiehormonen produceren zijn een uitdaging.

Gezien de recente catastrofale afname van palingpopulaties over heel Europa, is er niet veel tijd gegeven om deze vragen te beantwoorden en om het onvermijdelijk verlies van deze mysterieuze soort te voorkomen.

Conclusies: We kunnen concluderen dat Europese palingen erg efficiënte zwemmers zijn en dat gezonde, goed gevoede palingen in staat zijn de Sargasso Zee te bereiken waarbij ze genoeg energiereserves behouden voor reproductie. Lange afstand zwemmen van palingen en

5 Samenvatting de mogelijkheid te migreren worden negatief beïnvloed door infecties met virussen zoals EVEX. Ook PCB’s, welke uitgescheiden worden uit de gebruikte vetvoorraden na migratie, kunnen het energieverbruik en de stofwisseling negatief beïnvloeden. Dit kan resulteren in een verlaagd zuurstofverbruik, lagere glucose- en cortisolniveaus, een verminderde omzetting van aminozuren in de gluconeogenese en een verlaagde Basaal Stofwisselingssnelheid (SMR). Ofschoon we verschillende woonomgeving (habitat) factoren gevonden hebben die kunnen interfereren met de fitness en lange afstand zwemmen zoals virussen en PCB’s en die ten grondslag kunnen liggen aan de sterke afname van de palingpopulaties hebben we toch twee positieve resultaten gevonden die in de toekomst belangrijk kunnen zijn voor de reproductie van de paling onder kunstmatige condities. Allereerst hebben we gevonden dat zwemmen het proces van schier worden en een vroege maturatie in gang zet, Ten tweede hebben we voor de eerste keer bij hormoon-behandelde paling geobserveerd dat er afpaaigedrag in een groep optrad wat collectief en simultaan optredend was.

Dankbetuiging: "Onderzoek uit dit proefschrift werd gesponsord door STW project No. LBI66.4199 (Ir.J. van Rijsingen, Royaal BV, was sponsor in de gebruikerscommissie), De Europese Commisie (EELREP project QLRT-2000-01836), Het PCB experiment door EUROCHLOR (project officer drs. C. de Rooij), het vijverexperiment in Beesd door het Leids Universitair Fonds (LUF, 312/15-6-98/X,vT) en de GRATAMA-stichting (Harlingen, grant no. 9815). De vijver in Beesd werd ter beschikking gesteld door de Organizatie ter Verbetering van de Binnenvisserij (Nieuwegein, Directeur Dr. Lex Raat), de maturatie-experimenten door een subsidie van de OVB en LNV.

6 Summary

Simulated migration of the European eel (Anguilla anguilla L.)

Introduction: Over the past 25 years the population of European eel has been declining to such degree that major concerns have been raised for its long -term well being. Adult stocks have started to dwindle in the 1940's in major areas of the European continent, while recruitment (glass eel arrivals) has collapsed since the early 1980's. There is no sign of recovery and the phenomenon seems to occur over the natural range of the European eel (Anguilla anguilla L.). A parallel development is observed in the closely related American eel (A.rostrata) and the Japanese eel (A. japonica). The European eel (Anguilla anguilla L.) is a catadromic fish species with its spawning grounds thousands of kilometers away in the ocean. An important aspect of the reproduction of European silver eels is the huge distance they have to swim to reach their spawning grounds. After leaving the West European coast they still have to swim 5000-6000 km to the Sargasso Sea, the assumed spawning site. To cover this distance eels must swim continuously for 6 months at 0.5 Body Length per second, which requires an impressive swimming endurance capacity. Also high-energy reserves, coupled with low cost of transport are required. So, in addition it can be hypothesized that long term swimming capacity is a major prerequisite for reproduction. In this thesis we investigated the capacity of European eels to migrate over this distance. The freshwater phase of growth, sex-differentiation and 'silvering' a pre-adaptation to its ocean phase prior to migration will determine the quality of the spawners. This period in the fresh-water can cover a period of 5-50 years. Thus the habitat quality and habitat factors like shortage of food (leading to diminished fat stores), viruses and toxicants (e.g. polychlorinated biphenyls: PCB's) is important for the swimming fitness of the adults and quality of the gametes. In this thesis we will describe some of the topics of the life cycle of the European eel in order to understand more of the possible causes for decline of eel populations and factors that are involved in reproduction.

Freshwaterphase, orientation on the earth's magnetic field: In the literature several field studies in tanks, telemetric studies, studies with strong artificial magnetic fields, overriding the natural directional preference of eels, are indicative that orientation is accomplished through features of the earth magnetic field. Also the observation of magnetic substances in the skull and bones of eels strongly supports this view. We studied the circadian and monthly activity, the distribution patterns, and orientation to the earth’s magnetic field of yellow (non- migratory) female eels in a freshwater pond by means of microchips injected into their muscles. Detectors for microchips mounted in tubes were placed in the pond to detect if eels oriented themselves with respect to earth’s magnetic field. Based on the frequency of tube visits (search for shelter), the data indicated that the presence of eel in the tubes decreases gradually during the study period. We saw more activity during the night in the first months. There was a seasonal component in the orientation mechanism with a significantly lower preference component in the summer compared to the fall. A preference for tubes oriented in a south-southwest direction (the direction of the Sargasso Sea) in fall suggests an orientation to the earth’s magnetic field.

Freshwaterphase, silvering: The transformation of yellow (non-migratory) into silver eel (migratory) is called ‘silvering’, and takes place prior to migration. The mechanisms involved in the onset of 'silvering' of eels are largely unknown. Also a clear description of the different stages, which characterize this metamorphosis is lacking. Until recently silvering was, mainly

7 Summary

based on morphological parameters, split into two separate stages: 'yellow' and 'silver'. This classification did not take into account a possible preparatory phase. We described hormonal profiles of European eel during the silvering. We also used physiological parameters like body constitution and blood substrates. This transformation occurs in association with hormonal surges of testosterone (T), estradiol (E2), cortisol but not with those of thyroid hormones (TH) and growth hormone (GH) which have a maximum activity in spring and a minimum activity in summer and autumn. In contrast, cortisol levels in fall are elevated which play a role in mobilization of metabolic energy from body stores, to migratory activity and gonadal growth. Based on Principal Component Analysis with physiological, morphological and endocrinological parameters it is concluded that the transition is gradual and that eels go through several stages.

Freshwaterphase, role of thyroid hormone: For amphibians like frogs, the metamorphosis from larvae to adult is regulated by thyroid hormones. For other ecotherms like fish, also a role for thyroid hormone in metamorphosis is assumed like for salmon during parr-smolt transformation. However, from our year-cycle study we observed that thyroid hormones in eel are very high in spring but not in autumn during the 'silvering' (=metamorphosis) process. Therefore we can conclude that the thyroid hormones are possibly not involved in ‘silvering’. Another possibility is that their action is calorigenic and is involved in the control of the metabolic rate like in birds and mammals.. We measured overall heat production in free moving eels with different thyroid status with an accuracy of 0.1 mW by direct calorimetry. Hyperthyroidism was initiated by injection of T3 and T4 hormones while the effect of hypothyroidism was studied by exposing the animals to phenylthioureum. The results show for the first time at the organismal level, using direct calorimetry, that neither overall heat production nor overall oxygen consumption in eels is affected by hyperthyroidism. Therefore, we conclude that the thermogenic metabolism-stimulating effect of thyroid hormones is not associated with a cold-blooded fish species like the European eel.

The new type Blazka swim tunnel: We developed a Blazka swim tunnel of 127 liter with a total length of 2.0 meter and a length of the swimming compartment of 1.15 meter in order to test the endurance swimming capacity silver eels with a length of 80-90 cm. We applied the very accurate Laser-Doppler system to demonstrate the homogeneity of the flow in the swim tunnels. The actual flow was measured at different cross-sections and at different distances from the wall. A linear relationship was observed between the number of revolutions per minute of the motor and the measured water velocity. The linearity existed up to 0.9 m/s. The flow between 40-mm from the wall to the center stayed within a few percent of the setpoint. So, fish with a width of > 40-mm can not swim in the boundary layer. The eels used in the several studies needed an even wider space because of the amplitude of their tail beat. Furthermore we observed that the head of swimming eels remained between 50 and 100-mm from the wall.

Migration: Long term swim experiments over 5,500-km with virus-negative European eel demonstrates that eels are very efficient swimmers. Eels have a fat content of 10-28% with a mean of 20%, which is obviously the predominant energy store. 40% of the total fat reserve of silver eels is required for swimming 60% remains for development of the gonad. Animals with less than 13% fat would not be able to swim 6000-km. In comparison to other fish species like salmon, eels are very efficient swimmers with energy cost for migration that are 4-6 times lower than salmonids. The Cost of Transportation (COT) for eel was 0.68 kJ.kg-1.km-1 while the COT for trout was 2.73 kJ.kg-1.km-1. The estimated fat use for an adult eel to cross the Atlantic (6,000-km) would be 29% of its fat stores

8 Summary

corresponding to 58 g fat/kg eel while this would be for salmon 300 g/kg. At this moment it is not understood why eels are so efficient swimmers. In future studies hydrodynamics has to explain how does undulatory swimming (characteristic for anguilliform movement) work. Therefore two main questions have to be addressed: a) the topic of the muscle design: which muscle arrangement best suits the task of bending the body, b) how does the fish convert muscle power into swimming power.

Environmental effects on migration: Worldwide, eel populations have been dwindling over the last two decades of the previous century. The exact cause for this phenomenon is unknown, but possible causes include: PCB’s, viruses, and diminished fat stores. In order to study whether these factors had effect on the swimming performance and endurance of European eel, experiments were performed in 22 large swim-tunnels of 127 liter in the laboratory. PCB's: ). The results of our study revealed five major observations: First, PCB-exposed animals loose less weight and have lower glucose and cortisol (only swimming) levels compared to their unexposed controls. Second, PCB-concentrations on a lipid basis are 2.7 times higher in swimming compared to resting animals. Third, PCB-exposure significantly reduces oxygen consumption during swimming of the PCB-exposed animals from 400 km on -1 - (18 days) and this effect increases with time. The Cost of Transport (COT, [mg O2. kg . km 1]) is significantly lower in PCB exposed animals from 100 km up to 800 km. In addition the standard metabolic rate measured 2 days after the last swimming activity is significantly lower in the PCB-exposed animals. Fourth, the spleen is increased in the PCB-exposed swim animals but not in the PCB-exposed Control animals. Fifth, silver eels easily survive resting in marine water and forced swimming in fresh water, but not in a combination of these two stress factors. Plasma-pH, ion levels (sodium and potassium), plasma lactate acid, haemoglobin and hematocrit were unaffected by PCB-exposure. We conclude, that PCB- exposure interferes with the energy metabolism of silver eel in marine water and appears to interfere with cortisol control over (carbohydrate) metabolism. This effect was greater in swimming than in resting eel. Viruses: EVEX (Eel-Virus-European-X), HVA (Herpesvirus anguillae) and EVE (Eel Virus European) were detected in wild and farmed European eels (Anguilla anguilla) from the Netherlands, EVEX and EVE from farmed eels from Italy and EVEX from wild eels from Morocco. EVEX was also isolated from wild New Zealand eel (A. dieffenbachi). Elvers (A.anguilla) collected from eel farms in the Netherlands were mainly infected with HVA. Widespread infection of the eel-population with for instance EVEX virus may result from unlimited intercontinental eel transport. In addition, we show in large swim tunnels that eels infected with EVEX developed hemorrhage and anemia during simulated migration and died after 1,000-1,500 km. In contrast, virus-negative animals swam 5,500-km, the estimated distance to the spawning ground of the European eel in the Sargasso Sea. The virus-positive eels showed a decline in hematocrit, which was related to the swim distance. The virus- negative eels showed a slightly increased hematocrit. The observed changes in plasma of Lactate dehydrogenase (LDH), Total Protein and Aspartate aminotransferase (AST) are indicative of a serious viral infection. So virus infections and PCB's can possibly contribute to decline of eel populations.

Reproduction: We observed that in hormone treated European eel spawning behavior could be induced. This behavior of eels was collective and simultaneous corresponding to spawning in a group. This is the first time spawning behavior has ever been observed and recorded in eels and opens new perspectives for future research. Another important research result concerning the reproduction of this species was the observation that in 3 years old (juvenile)

9 Summary

European eels which swam for 173 days in Blazka swim tunnels, covering a distance of 5,500-km, at the end of the swim trial, the maturation parameters 11-ketotestosterone, pituitary levels of lutheinizing hormone (LH) and plasma levels of estradiol were higher (although not significantly) in the swim compared to the rest group. In contrast, the oocyte diameter was found to be significantly higher in the swim compared to the rest group. Based on these observations we conclude that a period of prolonged swimming might be a physiological stimulus necessary for the onset of maturation in the European eel. Experiments in future studies with adult virus free animals have definitely to prove that endurance swimming might be the natural trigger for gonadal maturation in European eel.

Panmixia, molecular work: The hypothesis that all European eel migrate to the Sargasso Sea for reproduction and comprise a single randomly mating population, the so called panmixia theory, was until recently broadly accepted. However, based on field observations, morphological parameters and molecular studies there are some indications that the Danish Biologist Schmidt's (1923) claim of complete homogeneity of the European eel population and a unique spawning location in the Sargasso Sea may be an overstatement. Recent molecular work (overview of different authors given in a review) on European eel indicated a genetic mosaic consisting of several isolated groups, leading to a rejection of the panmixia theory. Nevertheless, the latest extensive genetic survey of our Belgian colleagues from Leuven University indicated that the geographical component of genetic structure lacked temporal stability, emphasising the need for temporal replication in the study of highly vagile marine species. So, the Panmixia theory can still not be excluded nor confirmed.

Endangered species: During the last two decades of the previous century eel populations declined with 90-99%, possibly due to the synergy between human activities and oceanic fluctuations, making it an endangered species. For 3 million years the catadromous European eel succeeded in surviving and maintaining their characteristic life style with spawning areas in the ocean (possibly the Sargasso Sea) and its juvenile life phase of growth and sex differentiation in the freshwater at the European continent. The following recommendations can be made to prevent a total extinction of this species: a) Reduce fisheries pressure by establishing nature reserves in the Inland waters to conserve this territorial bounded fish species, b) Development of early warning systems at hydropower stations in order to protect a substantial part of the downstream migrating silver eels in fall during the migration season, c) PCB contamination should be monitored in all major hydro-systems and areas with low levels should be protected, d) To prevent the spread of parasites and virus infections international global instructions and sanitary standards for transportation of aquatic animals should be introduced, e) Research of artificial reproduction of the eel should be extended by focussing on administration techniques of hormones, more research is needed to understand the spawning behaviour of eels and the role of pheromones, and research should be focussed on understanding the natural trigger for reproduction. f) Molecular techniques like building in cells with genes (gentherapy), which produce maturation hormones, are a challenge. Considering the recent catastrophic decline of eel populations throughout Europe, not much time is given to answer these questions and prevent the irreversible loss of this mysterious species.

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Conclusions: We can conclude that European eels are very efficient swimmers and that healthy well fed eels are able to reach the Sargasso Sea leaving enough reserves for the reproduction. Endurance swimming of eels and the ability to migrate are negatively influenced by infections with viruses like EVEX. Also PCB's which are released from depleted lipid stores after migration may interfere with energy use and metabolism. This may result in reduced oxygen consumption, lower glucose and cortisol levels, a diminished conversion of amino acids in the gluconeogenesis and a reduced Standard Metabolic Rate. So, although we have found several habitat factors that may interfere with fitness and endurance swimming such as viruses and toxicants (PCBs) which could be a contributing factor to declining eel populations we found two positive results which in future studies may be of importance for reproduction of eels under artificial conditions. a) First, we found strong evidence that swimming triggers silvering and early maturation, b) Second, we observed for the first time in hormone treated eels that group spawning was collective and simultaneous.

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General Introduction: The European eel (Anguilla Anguilla L.) its lifecycle and reproduction; possible causes for decline of eel populations.

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Chapter 1

The European eel (Anguilla Anguilla L.) its lifecycle and reproduction; possible causes for decline of eel populations

1.1: Introduction, life cycle of the European eel: The European eel (Anguilla anguilla L.) is a catadromic fish species with its spawning grounds thousands of kilometers away in the ocean, possibly the Sargasso Sea. The life-history of the European eel (Anguilla anguilla L.) depends strongly on oceanic conditions; maturation, migration, spawning, larval transport and recruitment dynamics are completed in the open ocean (Tesch, 2003). Silver eels leave the continental rivers at different times, depending on lunar phase and atmospheric conditions. Then they are assumed to swim southward using the Canary and North-Equatorial currents and arrive six to seven months later at the Sargasso Sea to spawn and then die (Desaunay & Guérault, 1997; Tesch, 2003). The leptocephalus larvae are transported along the Gulf Stream and North-Atlantic Drift for a journey of six to nine months back to the eastern Atlantic coast (Lecomte-Finiger, 1994; Arai et al., 2000), where they metamorphose to glass eels. They ascent in this stage rivers and grow till partial maturity, six to fifty years later (Tesch, 2003). Other authors assume, however, that their journey until glass eel stage may last several years (van Utrecht and Holleboom 1985). During the last two decades of the previous century eel populations declined with 90-99% (figure 2) possibly due to synergy between human activities and oceanic fluctuations.

10000

1000

100

Ems 10 Den Oever Loire average other series

1 1950 1960 1970 1980 1990 2000 Year Figure 2: Trends in glasseel recruitment to the European continent. Reprinted from Dekker (2004) with permission.

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1.2: The oceanic phase The effect of oceanic currents on transport of leptocephali can be derived from indirect parameters like the influx of glass eel at Den Oever (The Netherlands) and the North Atlantic Oscillation Index (NAO-index). The NAO-index is the difference in air pressure between Portugal and Iceland. Knight (2003) discussed the correlations between the Den Oever glass eel recruitment index (DOI) and the North Atlantic Oscillation Index since 1938 and he found negative correlations (figure 1A). These can possibly be explained by the so called starvation and advection hypotheses: leptocephali survival could be affected by starvation and/or by unfavorable currents that prolong the duration of oceanic migration. Reduced transport rates due to reduction of current strength probably extend the period of migration, thus exacerbating the impact of low nutrition and exposing leptocephali longer to predation (Knights 2003). The same hypothesis is postulated for the recruitment of Anguilla japonica in relation to the El Nino-Southern Oscillation Index (Knight et al. 1996, Kimura et al. 2001). El Nino (Spanish word for 'male child' appearing around Christmas time and directing to the birth of Jezus Christ), initially referred to a weak, warm current along the coast of Ecuador and Peru which normally lasts only a few weeks to a month or more. However, every three to seven years, an El Nino event may last for many months allowing warmer waters of the western Pacific (area: China and Australia) to migrate eastward and eventually reach the South American Coast (Ecuador and Peru). This event may have significant economic and atmospheric consequences worldwide. It is also hypothesized that with the warming up of the earth, the Sub-Tropical Gyre (STG) warming inhibits spring thermocline mixing and nutrient circulation, with negative impacts on productivity and hence reduced food abundance for leptocephalus larvae (Knights 2003). From figure 1B we can see that recruitment of European glass eel rose in the warming periods of 1956-1962 and 1969-1977 to values well above the long-term average. During these periods the Sargasso Sea surface temperature anomalies at 100-250 m (SSTAs) did not show much fluctuations and were < 0° C (Figure 1B). Recruitment then declined markedly during the warming phase from the late 1980s, when SSTAs were consistently > 0 ° C (Knight 2003). So these correlations give strong indications that oceanic currents and temperature fluctuations can have its impact on the survival and recruitment of eel larvae.

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Figure 1A (Top): The Den Oever glass eel recruitment index (DOI, 5 year average, open circles) and the North Atlantic Oscillation Index (NAO-Index; 5 years Fast Fourier Transform average, solid circles) over 1940-2008). Modified from Dr. Brian Knights, University of Westminster, UK.

Figure 1B (Bottom): The ‘Sargasso Sea Surface temperature anomalies (ºC, Sssta) at a depth of 100-250 meters and the ‘Den Oever’ recruitment Index (DOI) (mean over 3 years, delayed with one year) over 1952-1995. ). Modified from Dr. Brian Knights, University of Westminster, UK.

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FRESHWATERPHASE

1.3: Freshwater phase: ecological information of eel populations and orientation on the earth's magnetic field. Ecological information about the home range and territorial behavior of eel in lakes and ponds is scarce. Also information about daily and annual activity patterns of eel is scarce. It is well known that eels have a strong homing behavior. When they are caught and transferred over 10- 200 km they return back to their earlier territories (Deelder & Tesch 1970, Hurley 1972). Research with tagged American eels (Anguilla rostrata) shows that their daily activity pattern is restricted to an area of 30-133 m. It was estimated that the home range (the foraging area) is restricted to 0.2 to 2,2 ha (Ford & Mercer 1986, Labar 1982). Eels show territorial behavior in the population. Helfman (1986) observed with a video-camera in wild American eel populations that large eel expel smaller eel from their territory. Aggression and hierarchy are only observed in low densities with large eel (> 400 mm) (Tesch 1977). At high densities there is less aggression. (After: de Nie 1988). Whether migratory animals can determine their global position by detecting features of the earth's magnetic field has long been debated (Gould, 1985, Walcott 1991). It is assumed that birds (Gould 1985, Walcott 1991), honeybees (Walker & Bitterman 1989), whales (Kirschvink et al. 1986) and dolphins (Walker et al. 1992) make use of this mechanism. For fish it is generally assumed that they are able to orient themselves on the earth’s magnetic field (Walker 1984) probably by sensors along the lateral line. So the question arises, do migrating silver eel orient themselves on the earth's magnetic field? For silver eel it is generally accepted that they migrate to the Sargasso Sea (Schmidt 1923, Miller & McCleave 1994, Fricke & Kaese 1995). It is likely that eels also use this mechanism to find its way to the Sargasso Sea. In the literature, several field studies with eel support the view that orientation is accomplished through features of the earth magnetic field. In tank experiments, Miles (1968) found that American silver eels orient themselves southwards a direction considered appropriate for the spawning migration to the Sargasso Sea. Telemetric tracking studies with European yellow and silver eels along the German North Sea coast indicated that the yellow eel preferred north-south axis while silver eels had a tendency towards a north-westerly direction (Tesch 1972, 1974). This direction for orientation was considered appropriate for European eels on spawning migration. In addition, strong artificial magnetic fields under laboratory conditions can override the natural directional preference of eels (Branover et al. 1971, Tesch 1974).

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Finally, strong evidence for orientation of eel on the earth-magnetic field comes from the observation that magnetic substances were found in the skull and bones of eels (Hanson et al. 1984).

1.4: 'Silvering', adaptation to its spawning migration During its life cycle the European eel (Anguilla anguilla L.) experiences two periods of metamorphosis. The first is the transformation from the planktonic marine stage (Leptocephalus larvae) into glass eel. This occurs near the coasts of Europe before entering fresh water. The second (partial) metamorphosis occurs after the juvenile growth and differentiation phase (> 4 years for males, >7 years for females) in the inland waters. Eels transform then from yellow eel into silver eel a process called ‘silvering’. During the latter transformation there is some proliferation of the gonads and an increase in eye size (Pankhurst 1982, Pankhurst & Lythgoe 1983). Furthermore, the body colour becomes silvery due to differentiation of pigment cells (Pankhurst & Lythgoe 1982); the alimentary tract shows regression, and the animal becomes fatter. These changes are part of the ‘silvering’ process, which precedes the spawning migration to the Sargasso, 6,000-km away from Europe. The mechanisms involved in the onset of ‘silvering’ of eels are largely unknown. Only one extensive study has been performed of the morphological and physiological characteristics at the different stages of eel silvering (Durif 2003). Based on principal component analysis (PCA) this author characterized some of the morphological and physiological parameters associated with silvering. Up to now silvering was split into two separate stages: 'yellow and 'silver'. This classification did not take into account a possible intermediate preparatory phase. Feunteun et al. (2000) classified eels into three stages: yellow, silver and yellow/silver. However, these stages were only based on external and visual variables (skin color, visibility of the lateral line and eye surface).

Seasonal, monthly changes over the year in parameters from fat metabolism, morphological, physiological and endocrinological parameters have never been described before for female eels from a brackish water population (Grevelingen lake, The Netherlands). We will determine which morphological, endocrinological and physiological characteristics are most altered during ‘silvering’. This will help us to understand better the transformation process of silvering. And whether these are adaptations for the migration phase in an oceanic environment.

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MIGRATION

1.5: Energy balance, the transatlantic migration Tucker (1959) suggested that the European eel is unlikely to perform the 6,000-km long journey to the spawning grounds. The adults would die in the continental waters due to depletion of the energy stores. According to this theory the American eel (Anguilla rostrata, LeSueuer) is the ancestor of both the European as well as the American eel. Morphologically there is a difference between the two species but he proposed that these differences were caused by environmental factors to which the larvae were exposed during their journey. Recently this hypothesis is rejected and it is proven, based on enzyme differences observed in larvae of both species that the American and European eel are different species (Comparini & Rodino 1980). Based on this information it is likely that the European eel has the energy supplies to perform the journey to the Sargasso sea and to develop gonads during this journey. Eel is a very fatty fish. During its life phase in the inland waters the animal stores fat in the body. Percentages of 27-29% have been measured in silver eel before onset of migration (Bertin 1956). The energy stores of fat are possibly used to provide the energy required for the journey to the spawning g- rounds. For salmon on the Fraser River, which have to swim a distance of 640 miles (1030 km) from the ocean upwards the river to the spawning grounds, it was observed that they performed this journey within 20 days. 96% of their fat supplies were consumed during this journey and 53% of their protein supplies (Brett 1965). Only for migrating salmon energy balance studies are known based on field data (Brett 1965, Brett 1972). Based on a theoretical approach it is suggested by the Danish scientists Boëtius & Boëtius (1980) that silver eel has sufficient energy supplies to swim 6,000-km. Their indirect approach is based on assumptions such as the energy costs for swimming (Schmidt-Nielsen 1972). It was always assumed that anguiliform swimming was much more inefficiently than (sub)carangiform swimming (see paragraph 1.6). It has to be definitely tested in the laboratory if European eels are capable to swimming under fasting conditions for 5,000-6,000 km on their initial energy stores.

1.6: Migration, hydrodynamics The swimming efficiency of migrating European silver eel during its spawning migration to possibly the Sargasso Sea, is to a great extent dependent on hydrodynamic parameters. Swimming styles based on body waves are classified on the basis of the proportion of the body during steady swimming contributing to thrust generation. The genus Anguilla

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lends its name to anguilliform locomotion, a purely undulatory mode of swimming, in which most or all of the length of the body participates and transfers thrust to water. Most fish species (subcarangiform, carangiform) swim with lateral body undulations running from head to tail. These waves run more slowly than the waves of muscle activation causing them, reflecting the effect of the interaction between the fish's body and reactive forces from the water (Wardle et al. 1995). In other fish species (subcaragiform, carangiform) the caudal fin is the main site of thrust production (Ellerby et al. 2000). In contrast, anguilliform swimmers show a high degree of body curvature during swimming. The body is long and thin; in eels it may be nearly cylindrical anteriorly, and somewhat laterally compressed towards the posterior. The caudal fin is small. A correlation exists to body curvature during swimming and the number of body segments. In undulatory swimming, a backward-travelling wave of bending is generated by the sequential activation of the segmental myotomes from head to tail. As the body bends, and the wave travels down the fish the body, the caudal fin pushes against the water generating a forward thrust (Altringham & Ellerby 1999). This forward thrust is mainly generated by the large amplitude that begins just behind the head and continues to the tail (Coughlin 2002). The side-to side amplitude of the wave is relatively large along the whole body, and it increases towards the tail. Fish that display different swimming modes commonly have different numbers of vertebrae. The European eel (A anguilla) has 110-119 vertebrae, the American eel (A.rostrata) has 103-110 vertebrae (Boetius 1980). Alternatively, the subcarangiform swimming rainbow trout (Oncorhynchus mykiss, Salmonidae) has 61-65 (Behnke 1992), and the thunniform swimming skipjack tuna (Katsuwonus pelamis, Scombridae) has 40-41 (vide review Coughlin 2002). High speed is not a characteristic of the pure anguilliform mode, most reports mention speeds around 0.5-1 BL/sec. For example American eels equipped with pressure sensing ultrasonic transmitters made frequent dives from the surface to the bottom during hours of daylight and darkness at speeds of 0.8-1.1 BL/sec. The maximum rate of ascent was 0.6-0.8 BL.sec (Stasko & Rommel 1974). Migrating Japanese silver eels (Anguilla japonica) have been tracked in the open ocean at a mean speed of 0.48 BL/sec (Aoyama et al. 1999). Low-tail beat frequency sustained swimming is powered by slow-twitch aerobic muscle. This slow-twitch muscle is present in eels as a wedge positioned along the lateral line (Ellerby et al. 2001). Until now it was assumed that anguilliform swimming is evidently less efficient in comparison to the carangiform mode of swimming. (Webb, 1975: Bone et al. 1995).

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This, however, seems to be in contrast to the enormous distance of several thousands of kilometers that eels have to swim to their spawning areas.

ENVIRONMENTAL EFFECTS ON MIGRATION

1.7: Concentration changes and possible toxicity effects of PCB's Organochlorine compounds were widely used after the second World War because they were cheap to produce and useful in many situations such as in agriculture for insecticides, in public health to control disease insect vectors and in industries for heat transfer fluid and plasticizers (Pelletier et al. 2002). It is estimated that over 30% of the one million tons of PCBs produced are still present in aquatic and terrestrial ecosystems (Borlakoglu et al. 1991). All organochlorines are very resistant to degradation and accumulate in the food chain because they are lipophilic compounds (Pelletier et al. 2002). PCBs (polychlorinated biphenyls), like other lipophilic, persistent pollutants, are mostly available to aquatic organisms from contaminated sediments in lakes and water courses, which may act as a source of PCBs to bottom-dwelling fish such as the eel (Tesch 1977). Fat percentages in silver eels reach up to 27 - 29%, before onset of migration (Bertin, 1956). So eels are very vulnerable for accumulation of PCB's in their fat stores. Larsson (1984) demonstrated that eels, which live and feed in direct contact with the sediment, might be exposed to higher amounts of PCB than fish in the open sea. And moreover, PCB concentrations were highest when eels were allowed to feed on benthic macroinvertebrates in the sediment, as a result of accumulation along the food chain (Hernandez et al., 1987). Many biomonitoring studies brought up evidence that PCB's massively accumulate in eel. Because eels have a very long juvenile phase up to 20 years in the contaminated inland waters they can accumulate PCB’s. Very high PCB levels were measured in the tissue of juvenile European eel originating from inland waters (Rahman et al., 1993; Haiber & Schöler, 1994; De Boer & Hagel.,1994; Hendriks et al., 1998). Total PCB concentrations in eel from rivers in north-western Europe range from 1.5 - 10 mg · kg-1 (De Boer & Hagel, 1994), therefore regularly exceeding the Dutch standards for human consumption, which is 5 mg · kg-1 of total PCB for eel. Another study by De Boer (1993) brought up that 85 - 90% of the toxic effect of the PCB in yellow eel is caused by PCBs 126, 156 and 118, which are mostly used for industrial purposes. The large monitoring study on PCB contamination by De Boer & Hagel (1994) found that the PCB levels in eel taken from the rivers Rhine and Meuse were amongst the

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highest values reported in freshwater fish from Europe. They reviewed other values from outside Europe, where the only higher total PCB values were reported by Sloan (1983). These values were 1500 - 4000 mg · kg-1 in 1977 in different fish species from the Hudson river, New York. PCB concentrations in eel from the Rhine have decreased substantially during the early eighties, but have been relatively constant since. Yellow eel has even shown to be a very practicable bio-indicator for the reflection of spatial differences and temporal trends of PCB contamination in fresh water. In addition, in an 8-year study by De Boer et al. (1994) it was demonstrated that elimination half-lives of PCB's are in the order of years. For the higher chlorinated CBs (chlorinated biphenyls) (hexa-octa-CBs), no elimination was found at all. The combination with a long juvenile phase in contaminated inland waters and the fact high fat content of eels, make that PCB's indeed accumulate in high quantities. From there the PCB's will be transported to the gonads and reproduction products. Also, since eel does not reproduce in inland waters, they do not lose parts of the accumulated PCBs. We will test when eels migrate and metabolize their PCB contaminated fat stores if this will have a negative impact on migration. We expect that PCBs released from fat stores during migration will negatively interfere with energy metabolism.

1.8: Effect of viruses on the swimming performance of silver eel A new factor that received little attention is the worldwide occurrence of viruses. For some fish species it is known that viruses can cause severe illness or even mortality when fish are under stressful conditions. For example in salmon the rhabdoviruses, Infectious Haematopoietic Necrosis Virus (IHNV) and Viral Haemorrhagical Septicemia Virus (VHSV), or the myxovirus, Infectious Salmon Anemia Virus, can affect haematopoietic tissues which leads to severe anemia (Wolf 1988). The most prominent cases of rhabdovirus infections in eel populations described in literature are infections with EVA (Eel-Virus-America) and EVEX (Eel-Virus-European-X (unknown)) which are serologically related (Kobayashi & Miyazaki 1996). Still, the role of rhabdoviruses in eel is largely unknown, and this study, were we simulated the migration of eel in the laboratory, may contribute for understanding its pathology. For viruses it is known they can affect the blood forming tissues, and typically become virulent during stress (Wolf 1988). Long-term migration of eel can certainly be considered as a stressful activity. Therefore one may assume that an outbreak of a virus infection can take place during this journey.

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REPRODUCTION

1.9: Maturation If silver eel are able to swim 6000 km this would be accompanied by enormous changes in body functions (Pankhurst & Lythgoe 1983) and body constitution. The transition from yellow eel towards silver eel is already accompanied by marked changes in morphology (Barni et al. 1985), body constitution (Lewander et al. 1974), fatty acids content (Dave et al. 1974) and to a lesser extent haematology (Johansson et al. 1974). The 6,000-km migration will result in even greater changes in body composition because the animals are starving, an enormous effort has to be performed and the gonads maturate. We therefore expect that the migration process might be important for maturation of the gonads. For the maturation of migrating silver eel several environmental stimuli are suggested like temperature (Boëtius & Boëtius 1967), light, salinity (Nilsson et al. 1981) and pressure (Fontaine & Fontaine 1985). Because eel migrate at great depth (Robins et al. 1979) it is suggested that high pressure would be the factor for synthesis of gonadotropines, the hormones that are responsible for ovarial stimulation (via oestradiol) for incorporation of vitellogenine from the liver in the oocytes (Selman & Wallace 1983, DeVlaming et al. 1984, Burzawa-Gérard & Dumas-Vidal 1991) and for maturation. This has been tested in a field-study by sinking off cages at a depth of 450 and 2000 m by Fontaine & Fontaine (1985). However these authors observed in this field-study no effects on the gonad maturation. The gonadotropin levels in blood also did not change (Fontaine & Fontaine 1985). From laboratory studies under high pressure, 2.5 MPa (Nilsson et al. 1981) respectively 101 atmosphere (Sebert & Barthelemy 1985, Simon et al. 1988) with eel, physiological changes were observed in the metabolism but no maturation of the gonads was observed. Even after a long term exposure to high pressure for a period of a month (Simon et al. 1988) respectively 4 months (Nilsson et al. 1981). Additionally, it has been revealed that hydrostatic pressure induces histotoxic hypoxia which is not a stimulating factor for gonad maturation (Sebert et al. 1993). Remarkably, never before the factor exercise as a potential stimulating factor has been examined. This could be an important stimulus for gonad maturation because enormous physiological and endocrinological changes are the result of exercise resulting in a change of body constitution. Characteristic for anadromic species like the Atlantic and the American salmon species, as well as for catadromic species like the eel, is that gonad development occurs in a period of great changes in body composition accompanied with heavy exercise. In this period marked changes in cortisol levels were observed (Butler 1968). At this moment it is not

22 Chapter 1

understood whether sexual maturation is accompanied with increased corticosteroid levels (review Idler & Truscott 1972, Pickering 1989). In several studies a correlation between sex hormones, body constitution and cortisol is demonstrated (Woodhead & Woodhead 1965, Mackinnon 1972, Wingfield & Grimm 1977). The possibility exist that cortisol is released when food intake is insufficient and energy stores are mobilized for exercise, gonad maturation, spawning or standard metabolism (Wingfield & Grimm 1977). Based on these observations it is suggested that a depletion of the energy stores is a prerequisite for gonad maturation (Fontaine 1961, Nilsson et al. 1981). This can of course not be the only factor because starving animals do not show gonad development (Boëtius & Boëtius 1967). In this study we will test the hypothesis whether the factor exercise and the resulting changes in body composition will initiate gonad maturation. To investigate this we plan to carry out these experiments under laboratory conditions in swim tunnels.

ANNEX 1:

Historical perspective of research at the reproduction of European eel The Scientist Antoni van Leeuwenhoek (1632-1723) originating from Delft (The Netherlands), has presented the results of his microscopically observations in letters. During that period in the 17e century there was a transition in Scientific approach in Natural and Biological Sciences, the Scientific Revolution. Experiment and resulting observation became of major importance and dogma's presented in books like the holy Bible were not taken for granted any more. Van Leeuwenhoek has performed a lot of research on the European eel (Anguilla anguilla L.) (Palm 1995, 1996) and in many cases he was inspired by Aristotle (384-322 BChr). During the time of Antoni van Leeuwenhoek the lifecycle of the European eel was unknown and he motivates why he finds it important to work on the reproduction of the eel (opening sentence of this Thesis). During the time period of van Leeuwenhoek there were two theories on the reproduction of the European eel: a) the 'generatio spontanea' (spontaneous generation), b) the 'viviparous' theory (delivering living young at birth). The first view stated that there was a spontaneous generation of eels from the mud, the Theory of the generatio spontanea (Intro: chapter 8).

23 Chapter 1

This was based on earlier thoughts of Aristotle written in his Historia Animalium VI, 15, "the eels come from what we call the entrails of the earth. These are found in places where there is much rotting matter, such as in the sea, where seaweed’s accumulate, and in the rivers, at the water's edge, for there, as the sun's heat develops, it induces putrefaction." (Bertin 1956). Another indication for a generatio spontanea was that eels may originate from dew in the month May (Intro: chapter 9). A third version of the generatio spontanea describes the originating of eels from the stripped skin of eels, the so called 'Aels-vellen' (Intro: chapter 11). This passage in his letters about the 'Aels-vellen' also originates from Aristotle (Historia Animalium, VI, 15). “We have seen eels emerging from the skins of these worms; and if one tears the worm apart and opens them, one sees clearly inside them” (Bertin 1956). The other theory of the origin of eels was that they were viviparous (giving living young at birth). At the Intro (chapter 10) in this thesis Antoni van Leeuwenhoek describes that when he opens the abdomen of eels that he find little eels. Possibly these were parasites (worms), but all these passages from his letter show that Antoni van Leeuwenhoek was curious and intrigued by the origin and reproduction of the European eel. Passages from the letters of Antoni van Leeuwenhoek were selected in this thesis for illustration at the back of some chapter intro-pages. Antoni van Leeuwenhoek had around 1700 AD his microscope to study the physiology of eels. We used in this thesis modern tools like radio-immuno-assays for hormones, enzymatic assays for substrates, swim tunnels calibrated with Laser-Doppler techniques for energy balance studies, electronic tagging and monitoring techniques for orientation on the earth's magnetic field, indirect and direct- calorimetric techniques to measure overall metabolic rate, bomb-calorimetry techniques to measure energy content of carcasses, electron microscopy techniques for virus detection, and a Bioanalysis technique using a reporter gene assay based on activation of luciferase (CALUX) to measure PCB's. With these tools we were able to elucidate the life history of this mysterious animal already in much greater extent than Antoni van Leeuwenhoek. In future studies we hope that modern molecular techniques and animal tracking technique will even further help us to unravel the mysterious life cycle of the eel.

24 Chapter 1

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AIMS AND OUTLINE OF THE THESIS

|The objective of this study was to elucidate this oceanic phase of migration for the European eel (Anguilla anguilla L.) in the laboratory. In order to investigate this we simulated the migration of spawners in the laboratory by building 22 large swim tunnels of 127 liter specially developed for long term migration. Therefore the title of this thesis is: 'Simulated migration of European eel'.

What matters is how much energy is required for the crossing of the Atlantic Ocean. The second aims of the investigations were to study environmental factors that may have impact on the migration capacity like viruses and pollutants (PCB's). The third aims of the investigations were to test whether endurance swimming induces sexual maturation and development of the gonads.

This thesis consist of four major parts: A) Preparation to migration B) Simulated migration C) Effect of environmental factors (viruses and PCB's) on the migration D) Effect of swimming on maturation.

In chapter 1 we will give an introduction to the thesis and a general introduction to the investigations. In chapter 2 we studied the circadian and monthly activity, the distribution patterns, and orientation to the earth's magnetic field, of yellow (non-migratory) female eels in a freshwater pond by means of microchips injected into their muscles. Detectors for microchips mounted in tubes were placed in the pond to detect if eels oriented themselves with respect to earth's magnetic field. We tested the hypothesis whether there is a preference for tubes oriented in a south-southwest direction (the direction of the Sargasso Sea) in the fall suggesting an orientation to the earth's magnetic field. Based on these observations we decided to position the 22 Blazka swim tunnels in the direction of the Sargasso Sea (figure 1).

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Figure 1: 22 Blazka swim tunnels of 127 liter positioned in the direction of the Sargasso Sea.

In chapter 3 we described morphological and metabolic parameters and in chapter 4 endocrine profiles of European eel during the process of 'silvering' the transformation of yellow (non-migratory) into silver eel (migratory), prior to migration. We tested the hypothesis whether yellow and silver are two clear separated transition forms or that this transition is gradual and that eels go through several development stages. Because we observed in chapter 4 that 'silvering' is accompanied with hormonal surges of testosterone (T) and estradiol (E2) but not with thyroid hormones (TH) which have a maximum activity in spring and a minimum activity in summer and autumn we studied in chapter 5, the overall heat production in free moving eel with different thyroid status by direct calorimetry. We wanted to test whether the action of this hormone is calorigenic and involved in the control of metabolic rate. In chapter 6 we described the development and calibration with the Laser- Doppler technique of the 127 liter Blazka swim-tunnels for simulated migration in the laboratory. In chapter 7 we tested the hypothesis whether substrates (FFA, glucose), the stress hormone cortisol, parameters from the ionic balance (sodium, potassium, chloride) and lactic acid were affected by different swimming speeds up to 3.0 Body Lengths per second. We wanted to investigate if a swimming eel remains in homeostasis for physiological and endocrinological parameters in the blood plasma at different swimming speeds. In chapter 8 we gave an estimation of the energy required to cover the 6,000-km distance. This corresponds to 120 g per kg (12%) or 40% of the initial fat reserves. In chapter 9 we tested the efficiency of anguilliform swimming in comparison with (sub)carangiform.

33 Chapter 1

Until recently it was assumed that anguilliform swimming was less efficiently than (sub) carangiform swimming. Our results give new insights in this matter and demonstrate that anguilliform swimming was 4 to 6 times more efficient than non eel-like fish. In chapter 10 we wanted to test the hypothesis that eels infected with the rhabovirus EVEX (Eel Virus European X)-virus, developed hemorrhage and anemia during simulated migration in large swim tunnels, and died after 1,000-1,500 km. In contrast, virus-negative animals swam 5,500 km, the estimated distance to the spawning ground of the European eel in the Sargasso Sea. In chapter 11 we wanted to test the hypothesis if eel viruses in eel species from various geographic regions are widespread. We isolated from three eel species from various regions several viruses and demonstrate that eel viruses are worldwide widespread among the eel populations. In chapter 12 we studied the effect of PCB’s on oxygen consumption, weight decline, plasma-pH, ions (sodium, potassium), lactic acid, hemoglobin and hematocrit during a simulated migration over 750-km. We wanted to test the hypothesis whether there was a weight loss, reduced oxygen consumption and lowered glucose and cortisol levels due to PCB exposure. In chapter 13 we wanted to test whether a swim trial of 5,500-km over a six month period induced gonad maturation. Finally in chapter 14 we give recommendations for protection of eel populations and suggestions for future research. In Annex 1 we give an overview of the literature on the lifecycle, evolution and reproduction of the European eel and discuss if the Sargasso Sea is the only spawning area of the European eel. In Annex 2 we studied the reproduction process of eels without using swim tunnels. Gonadal development and spawning behavior of artificially-matured European eel (Anguilla anguilla L.) was studied by giving animals hormone injections. This is the first time group spawning behaviour has ever been observed and recorded in eels.

The research described in this thesis was carried out in two postdoctoral projects: by a grant of the Technology Foundation (STW), project no. LB166.4199 and by the European commision (Project QLRT-2000-01836, EELREP). Smaller grants were given by EUROCHLOR for PCB-work, Gratama-LUF (grant. No. 9815) for pond experiments and Organization for Improvement of Inland Fisheries (OVB) and LNV for maturation experiments by treating animals with hormones.

34 Chapter 2

Microelectronic detection of activity level and magnetic orientation of yellow European eel, Anguilla anguilla L., in a pond

V. van Ginneken1, B. Muusze1, J. Klein Breteler2, D. Jansma1, G. van den Thillart1

1) Integrative Zoology, Institute of Biology Leiden, van der Klaauw Laboratorium, P.O.Box 9511, 2300 RA Leiden, The Netherlands ( e-mail: [email protected]). 2) Organization for Improvement of Inland Fisheries, Postbus 433, 3430 AK Nieuwegein, The Netherlands

Keywords: circadian rhythm, migration, eel tracking, micro electronic detection, activity patterns, geomagnetism

Published in: Environmental Biology of Fishes (2005) 72: 313-320

35 Chapter 2

Onderscheid tussen aal en paling Onder de visschen die onse rivieren of wateren voort brengen, kan ik maar twee soorten van visschen die men seijt dat geen schobbens hebben, de eene soort wort alhier genoemt Ael en Paling, en in andere steden wertse wel alleen met den naam van Ael genoemt. Dog men maakt alhier groot onderscheijt inde selvige, om dat de paling veel vetter en aan genamer van smaak is, en dier halven veel dieren wert verkogt. De tweede soort word genoemt Puijt Ale de laastse sijn kort en dik en seer weijnig. (Antoni van Leeuwenhoek, Brief No. 81 [42], 25 juli 1684).

Chapter 2

Microelectronic detection of activity level and magnetic orientation of yellow European eel , Anguilla anguilla L., in a pond

Synopsis

We studied the circadian and monthly activity, the distribution patterns, and orientation to the earth's magnetic field, of yellow (non-migratory) female eels in a freshwater pond by means of microchips injected into their muscles. Detectors for microchips mounted in tubes were placed in the pond to detect if eels orientated themselves with respect to earth's magnetic field. Based on the frequency of tube visits (search for shelter), the data indicated that the presence of eel in the tubes decreases gradually during the study period. We saw more activity during the night in the first months. There was a seasonal component in the orientation mechanism, with a significantly lower preference component in the summer compared to the fall. A preference for tubes orientated in a south-southwest direction (the direction of the Sargasso Sea) in fall suggests an orientation to the earth's magnetic field.

Introduction

Anguillid eels have a complicated life cycle, which takes place partly in freshwater, and partly in seawater. Little is known about this cycle, particularly the ecology or behavior of the eels during the oceanic phase. Based on the work of Schmidt who caught leptocephali (the larvae of the eel) in the ocean, it is assumed that the spawning grounds of the European eel are 6000 km away in the Sargasso Sea (Schmidt 1923, Miller & McCleave 1994, Fricke & Kaese 1995). Transport of the leptocephali larvae by the sea currents, towards the coasts of Europe, probably lasts 1 - 3 years. It is probably not purely a passive process (Lecomte-Finiger 1994). On reaching the coasts of Europe, the larvae transform into glass eel. They can be observed in March - April in the North Sea and in July in the Baltic Sea. When they invade the inland waters they develop pigmentation (Tesch 1977) and are called yellow eel. This is the juvenile life phase of feeding and growth. Gonad differentiation occurs during the time spent in fresh water. After this growth period, which last 3 - 12 years in males and 5 - 35 years in females, the animals prepare themselves for their return journey to the ocean. An enlargement of the eyes, a regression of the digestive tract and a silvering of the body color characterize this phase. However, little ecological information is available about this freshwater phase of several years prior to migration. Processes like circadian rhythm, annual

36 Chapter 2

activity patterns, hierarchy, foraging area and distribution patterns of eels in relation to season and age, and orientation on the earth's magnetic field need to be elucidated. The present work mainly concerns the observation of activity patterns of 40 female eels by means of microchips on a 0.8 ha pond during the first 7 months of the 2½ year field research period. The basis for the experimental set up with the tubes with electromagnetic detection is the behavioural response of eels to search for shelter. Probably this behaviour can be explained by the eel’s vulnerability to predation in the shallow fresh water. Another possible explanation is that it is a way to protect itself against harmful environmental factors or a way to conserve energy (Edel 1975). The latter factor can be explained because an increase of activity is observed with decreasing shelter availability. This was indicated by Edel (1975) with the term “ negative skiasmokinesis” (skiasma=shelter, shade). Therefore, based on this behaviour, they will visit the tubes with microchip detectors (Figure 1). In this way the frequency of ‘tube visit’ (search for shelter) and preference position of eels in every tube can easily be measured, not only over the course of one day, but also over the seasons. In order to investigate if eels oriented themselves on the earth's magnetic field, the tubes in the pond were positioned in an alternating arrangement, in the direction of the Sargasso Sea (south-south-west), or opposite to it (west-north-west, see Figure 2). So, orientation on the earth's magnetic field can be investigated depending on the season. This study will give information about the activity patterns and orientation in relation to the earth's magnetic field of European eel at the end of the fresh water period before the migration period in the ocean starts.

Material and methods

In June 1999, we placed 48 PVC tubes, with an inner diameter of 4.3 cm and a length of 80 cm, in a 1.5 m shallow pond of 0.8 ha (95 x 85 m). We mounted detectors for microchip transmitters on the tubes (Figure 1). The detector consists of a printed circuit board (Trovan type LID656) in a waterproof box, and a detection antenna. The antenna is a solenoid coil of 0.23 mH: 225 windings over a length of 67 cm and with a diameter of 7.5 cm. The coil is placed between the double layers of the PVC tube. All 48 detectors are individually linked by a waterproof cable (PUR-CY6x0.25) to 48 serial interfaces (Com ports) of a computer. Special software was developed to record all activities. We connected the registration computer near the pond in Beesd (the Netherlands), to the PC network system of the University of Leiden (using PC- Anywhere software).

37 Chapter 2

Figure 1: PVC-tube with Trovan detector (Trovan type LID656) in a waterproof box and a detection antenna. The antenna is a solenoid coil of 0.23 mH: 225 windings over a length of 67 cm and with a diameter of 7.5 cm. The coil is placed within the double skin of the PVC tube. All 48 detectors are individually linked by a waterproof cable (PUR-CY6x0.25) to 48 serial interfaces (Com ports) of a computer.

We placed the tubes with the detectors (Figure 1) in the pond according to a chessboard pattern (Figure 2). Twenty-four tubes were oriented in the direction south-south-west at 202.5o (direction of the Sargasso Sea), and 24 tubes were oriented in the direction west-north-west at 292.5o (perpendicular to the direction Sargasso Sea, Figure 2). On 2 June 1999, the pond was stocked with 26 eels. On 21 July, 1999 we placed 14 additional eels into the pond. The eels were obtained from a hatchery (Royaal BV) with a mean age of 2 years, a mean weight of 578 ± 90 grams and a mean length of 64 ± 4 cm. We injected a Trovan ID 100 implantable transponder microchip (2.1 x 11.5 mm) in a biocompatible glass capsule in the dorsal muscle 10 cm behind the head of every eel. These transponders are passive transmitters that transmit an ID code when activated in an electromagnetic field of 128 kHz.

38 Chapter 2

Figure 2: The tubes with the detectors were placed in a pond of 0.8 ha (95 x 85 meter) according to a chessboard pattern, 24 tubes were orientated at 202.5o (direction of the Sargasso Sea), and 24 tubes were orientated at 292.5o (opposite direction).

39 Chapter 2

Table 1: Fish occupation of the pond in May 1999 at the start of the experiment.

Length Total kg Carp Cyprinus carpio 25-40 cm 50.0 Bream Abramis brama >35 cm 75.0 Roach Rutilus rutilus >15 cm 25.0 Rudd Scardinius erythrophthalmus >13 cm 10.0 Zander Sander lucioperca >45 cm 3.0 Eel Anguilla anguilla (our tagged eels) >57 cm 22.5 Fry Unspecified fish brood < 8 cm 10.0

The Trovan-system continuously recorded all eel activity in the pond. These data were translated to migration and distribution patterns of the individual eels in the pond. The precise distance between the various tubes is known, so an indication of the distance that eels migrate can be recorded. In principle, this record is the minimum distance an eel has migrated. Every tube has a capture device. The computer records directly which tube is occupied by which eel. Every 6 months we captured the eels via the capture devices for ‘on site’ blood sampling. The eels were anaesthetised (100 p.p.m. benzocaine) and released again directly after blood sampling. Blood samples (1.5 ml) were tested for hormone levels at a later stage. This information will be combined with maturity and activity measurements in a later analysis. We chose the density of eel in the pond (24 kg eel 0.8 ha-1 or 1 eel per 250 m3) so that the pond can produce enough natural food for growth (Klein Breteler et al., 1990). We stocked the pond with 163 kg of other fish species and fry (Table 1). We expected that maturation of the eel would be possible during the 2 years following initial stocking.

Calculations and statistics: We defined the maximal degree of occupancy (100%) as the total number of hours per month where all 48 tubes are fully occupied. For instance for a month with 31 days the maximal occupancy degree is 3 5712 hours (31 days*24 hours*48 tubes=100%). In order to investigate if eels oriented themselves on the earth's magnetic field, the tubes in the pond were positioned in an alternating way, in the direction of the Sargasso Sea (south-south- west), or opposite to it.

40 Chapter 2

For every individual eel, the seasonal component in the orientation mechanism has been calculated following:

number of hours in south-south-west tubes Preference index = ------(per eel) number of hours in (south-south-west) + (west-north-west tubes)

According to this index: 1: indicates 100% preference for south-south-west tubes, 0.5: indicates an undirected preference 0: indicates a 100% preference for west-north-west tubes.

The summed values of the orientation indices of all eels are expressed per month in the orientation-coefficient. Our 'between individual months analysis' for the preference index did not come up with a clear significance below 0.05, but often bordering this value. The data however showed a trend with higher values in the fall compared to the summer months. Therefore we pooled our data over the summer period (June, July, August) vs. fall (September, October, November). We applied a one-way ANOVA, comparing this summer period with fall. P≤ 0.05 was considered statistically significant. Normality of the data and homogenity of

variances were checked by Kolmogorov-Smirnov and Fmax tests

Results

Immediately after the first 26 eels were released in the pond (2 June at 20:00 hours) they searched for shelter in the tubes. Only 20 min after being released, the first eel (code: 0001FC39DB / Saskia) was detected in tube 40 (position E6), a southwest oriented tube. Some eels stayed for a long time uninterrupted in one tube. For example, one eel (code: 0001FCDBB9 / Hanneke) entered a tube on 17 June and left 2 months later on 16 August. Since the total occupancy remained at about 12.5% for the first month, we decided to increase the number of eels from 26 to 40. After releasing the second group of 14 eels (July 21 at 20:00 h) they did not visit the tubes until the next morning. The first eel from the second group (code: 0001FCAF0D / Louise) entered at 7:47 h tube 34 (D8), a northwest orientated tube that was not yet occupied

41 Chapter 2

by another eel. Hereafter, in the coming hours or days, the eels of the second group entered tubes that were not yet occupied by other eels of the first or second group. Presence of eel in the tubes, which can be derived from the total time of eels in the tube, decreases during the period June-November 1999. After the first month there is an increase of the presence of eel in the tubes, due to the extra 12 eels we put in the pond. In July, 23.8% of the tubes were occupied but in November only 6.0%. In the event that all the eels would find shelter in the tubes during the daytime, then 41.7% occupancy should be found. During the analysed period the average occupancy was 13.7%. Figure 3 gives an overview of the seasonal division of eels over the pond. In summertime (June, July, August) the eels are equally divided over the pond. In autumn the tubes along the edge of the pond are more occupied. There was a circadian activity pattern, with activity during day and night (not depicted) during the first months an increased activity during the night (between 19:00 and 08:00 h). In July for example during daytime 27% of the tubes were occupied, while during nighttime only 16% were occupied. In November the circadian activity pattern was less clear, partly because of the low presence of eel in the tubes. During winter the water was colder and the eels apparently prefer to stay in the mud on the bottom of the pond. As an example, we described the activity pattern for one eel (0001F85D9D / Floortje) for August 1999. In this month we registered, based on our detection method with tubes, that this eel swam at least 609 meters between the tubes with a minimum average speed of 20 cm-1. The animal started in the middle of the left part of the pond (tube C8), swam to the south-east (tube F4) returned to the left part of the pond (tubes A-F: 8 and 7) and ended in the outer south- western part (tube F2) of the pond. In principle, because the tubes serve as marking points, reflecting the minimal covered distance, the real distance, and maximum speed that the eel swam, can be much higher. During the period June to November 1999 there is an increase in preference for the south-south-west (the direction Sargasso Sea) oriented tubes. So eels have a preference on the earth's-magnetic field for south-south-west (202.5o) oriented tubes. The preference component was 0.4 in June increasing to 0.68 and 0.67 in September and October respectively. Comparing the summer months (June, July, August) with fall (September, October, November) resulted in a significant higher Preference Index during fall (P ≤ 0.045).

42 Chapter 2

Figure 3: For eel it is observed they are seeking regularly for shelter, so they will visit the tubes with microchip detectors. In this way the frequency and preference position of eels for every tube can easily be measured, not only over a day, but also in relation to season. Y-axis: denotes 'Total time of eels in the tubes per month' in [hours]. A: June, B: July, C: August, D: September, E: October, F: November in 1999.

A B

600 600 500 500 400 400 300 300 200 200 A B A 100 C B

100 D C 0 E D 0 F E 2

F 4 2 6 4 8 6 8 C D

600 600 500 500 400 400 300 300 200 200 A B A 100 C B

100 D C 0 E D 0 F E 2

F 4 2 6 4 6 8 8 F E

600 600 500 500 400 400 300 300 200 200 A B A 100 C B

100 D C 0 E D 0 F E 2

F 4 2 6 4 6 8 8

43 Chapter 2

Discussion

The basis for the experimental set up with the tubes with electromagnetic detection is the endogenous behavioural response of eels to search for shelter. The circadian rhythms found in our study indicate that the eels are more active during darkness than during day light. This was also observed by Edel (1975, 1976) and given the name “endogenously scotokinetic” (scoot= dark). The animals showed increased nocturnal activity with crepuscular peaks of activity corresponding with transitions from light to dark and vice versa (Edel 1976). From literature, it is known that activity patterns were also dependent on the maturational stage of the animals. Immature eels were more nocturnally active and showed peaks of activity at light-dark transitions. This was also observed in this study with yellow eel. In contrast, maturing eels were equally active by day and by night but remained responsive to light-dark transitions (Edel 1976). Hain (1975) reported that yellow eels have several ‘try outs’ or dry runs before their final migration to the Sargasso Sea as silver eels. A temporary slight maturation of yellow eels can possibly cause the observed differences in activity patterns during the season, with a decrease as the migration season progresses in the fall. When the animals become mature they are nocturnal and overall activity decreases. Besides a maturing effect, the seasonal effect can also be explained by temperature changes. In November the tubes are less occupied. Probably the eels hibernate and burrow themselves in the mud in order to reduce the contact with the environment (Walsh et al. 1983). Hibernation or metabolic depression has recently also been demonstrated for European eel in a micro- calorimeter under conditions of anoxia. The eel (mass: 125 g) reduced its metabolic rate to 30% of the standard metabolic rate (SMR) while no lactate-ethanol conversion has been observed (van Ginneken et al. 2001). This may be an important survival strategy to save energy stores and diminish end-product accumulation (Ultsch 1989). The eels we released on the pond were the first year yellow (non-migratory). For yellow eel it is known that they have a very low drive for migration or long distance journeys. This is interesting because Gunning & Shoop (1962) found their territory is restricted to 61 linear meter or less. Research with tagged American eel, Anguilla rostrata, shows that their daily activity pattern restricts itself to an area of 30-133 m. Their territory or home range, which is defined as the foraging area of an eel which it daily occupies, restricts itself to 0.2-2.2 ha (Labar 1982, Ford & Mercer 1986). In our study, the route the individual eel (0001F85D9D/Floortje) swam in August covered nearly the whole pond of 0.8 ha.

44 Chapter 2

It is remarkable, that in our study a seasonal component has been observed in the orientation mechanism of yellow eel. A seasonal component was also observed in yellow eel in the study of Hain (1975). When these eels were given the choice between swimming upstream (positive rheotaxis), downstream (negative rheotaxis), and no current (neutral response), the animals displayed in August an equal response for all choices. But two months later in October during the migratory season, a strong negative rheotaxis was observed (Hain 1975). The author explains this result with the suggestion that yellow eels have several 'try outs' or 'dry runs' several years prior to their final return journey as silver animals to the Sargasso Sea. After each ‘false start’ or ‘trial run’ the migratory characteristics will again decrease, either totally or to a large degree, until the next migratory season (Hain 1975). The observed differences in rheotaxis between August and October found by Hain (1975), or the observed differences in the preference component in our study, are indicative for a seasonal dependent orientation on the earth's magnetic field. This can possibly be explained by this theory of migratory ‘try outs’ of yellow eel. By using tubes that were laid out following a chessboard pattern, alternatively in a south- south-westerly or a west-north-westerly direction (90° difference), we were able to study the orientation behavior of sub-adult eel during the whole season, which covered a period of 7 months. We have to admit that our electronic system was not capable of distinguishing a 180 ° difference in orientation of the eels when inside the tubes. In addition, we can not observe within the same tube whether an animal is with its head in SSW vs. NNE direction; the same for the 90 °opposite tube: WNW vs. ESE direction. This is the topic of the so called "directional ambiguity". We are aware of the limitation of our method on this point but are strengthened in our opinion that yellow eels have a preference for SSW tubes (direction Sargasso Sea) in the fall by the following two observations. First, Hain (1975) also observed for yellow eel a strong negative rheotaxis in fall. Secondly, we observed the same pattern in Preference Index (preference SSW tubes in fall) the following two consecutive years (1999 and 2000) in the same pond with the same experimental set up and animals. In fall 1999, the Preference Index was significantly higher compared to the summer period (P≤ 0.045). Also in fall 2000, a significant higher Preference Index was observed compared to the summer period (P≤ 0.038) (unpublished results). The possibility exists that homing of eels is based on olfactory principles. However results in the Baltic with tagged anosmic eels (the olfactory organ has been removed) exclude this mechanism. No difference in orientation was observed with a control group during a 100-

45 Chapter 2

500 km migration (Karlsson 1984). Another possibility for eel to determine their global position is by detecting features of the earth's magnetic field. Many animals in nature, like birds (Walcott 1991), honeybees (Walker & Bitterman 1989), whales (Kirschvink et al. 1986), dolphins (Walker et al. 1992), loggerhead turtles (Lohmann & Lohmann 1996), and possibly also fish (Walker 1984) make use of features of the earth's magnetic field like the magnetic field intensity and the magnetic inclination angle. In fish lateral line organs may be important (Walker 1984) For sockeye salmon (Oncorhynchus nerka) fry and smolt it has been demonstrated they use both celestial and magnetic cues as orientation mechanism when migrating to and from nursery lakes, respectively (Quinn 1980, Brannon et al. 1981, Quinn and Brannon 1982). The directional preferences were innate and population specific depending on characteristics of the waters the fish grew up. In the literature, several field studies with eel support the view that orientation is accomplished through features of the earth magnetic field. In tank experiments, Miles (1968) found that American silver eels oriented southwards a direction considered appropriate for the spawning migration to the Sargasso Sea. Telemetric tracking studies with European yellow and silver eels in the German North Sea coast indicated that the yellow eel preferred a north-south axis while silver eels had a tendency towards a north-westerly direction (Tesch 1972, 1974). This direction for orientation was considered appropriate for European eels on spawning migration. In addition, strong artificial magnetic fields under laboratory conditions can override the natural directional preference of eels (Branover et al. 1971, Tesch 1974). Finally, strong evidence for orientation of eel on the earth-magnetic field comes from the observation that magnetic substances were found in the skull and bones of eels (Hanson et al. 1984). In conclusion, using this elegant method with tubes positioned according to a chessboard pattern in a pond, we demonstrated that the preferred orientation along the earth's- magnetic field of yellow eel, during sheltering in the tube, is season-dependent. Advantages of this method are no handling stress of the animals, measurement of the position preference of a large group, and the fact that the animals were in their natural environment.

Acknowledgments

We thank Dr.Lex Raat, Organization for Improvement of Inland Fisheries, Nieuwegein, the Netherlands, for supporting this project and providing the pond, Frans Jacques for technical assistance and pond management, and Royaal BV for providing 48 female eels. Technical assistance was provided by Rob van der Linden, Rinus Heijmans, Ab Gluvers, Jeroen Mesman,

46 Chapter 2

Frits van Tol and Gerard Kostense. Technical detection equipment on the pond was subsidized by a grant of 'het Leids Universitair Fonds' (LUF, grant no. 312/15-6-98/X,vT) and the GRATAMA-foundation (Harlingen, grant no. 9815). The eel migration project at the University Leiden is supported by a grant of the Technology Foundation (STW), which is subsidized by the Netherlands Organization for Scientific Research (NWO), STW-project no. LBI66.4199. The field experiment was also supported by the EU EELREP project no. Q5RS- 2001-01836.

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Walker, M.M.& M.E. Bitterman. 1989. Honeybees can be trained to respond to very small changes in geomagnetic field intensity. Journal of Experimental Biology 145: 489-494.

Walker, M.M., J.L. Kirschvink, G. Ahmed & A.E. Dizon. 1992. Evidence that fin whales respond to the geomagnetic field during migration. Journal of Experimental Biology 171: 67- 78.

Walsh, P.J., G.D. Foster.& T.W. Moon. 1983. The effects of temperature in metabolism of the American eel (Anguilla rostrata, Le Sueuer) compensation in the summer and torpor in the winter. Physiological Zoology 56: 532-540.

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SILVERING OF EUROPEAN EEL (Anguilla Anguilla L.): SEASONAL CHANGES OF MORPHOLOGICAL AND METABOLIC PARAMETERS

V. van Ginneken1; C.Durif2; S. P. Balm3; R Boot1; K. M.Verstegen4; E.Antonissen1; G.van den Thillart1.

1) Integrative Zoology, Institute Biology Leiden (IBL), van der Klaauw Laboratorium, P.O.Box 9511, 2300 RA Leiden, The Netherlands. 2) Cemagref, Unite Ressources Aquatiques Continentales, 50 avenue de Verdun, 33612, Cestas, France 3) Animal Physiology, Department of Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. 4) Department of Animal Nutrition, Wageningen Agricultural University, Marijkeweg 40, P.O.Box 338, 6700 AH Wageningen, The Netherlands.

Corresponding Author: Dr.V.J.T.van Ginneken, Integrative Zoology, Institute Biology Leiden (IBL), van der Klaauw Laboratorium, P.O.Box 9511, 2300 RA Leiden, The Netherlands, FAX: +31(0)71-5274900, E-mail: [email protected], TEL: +31(0)71-527749

Submitted to: Animal Biology

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Over de giftigheid van palingbloed Hoorende een Viscooper (die doende was met een groote Palingh het Vel af te halen) seggen datmen sich wel most wachten, dat het bloet van een Palingh, niet in de Oogen quam, om dat het ongelooffl. doodel. pijn veroorsaeckte, die een gantsche dach duerde, sonder datmen het Oogh na behooren konde gebruijcken. Ick heb aenstonts ses Palingen gecocht, omme was het mogelijck, de redenen vande groote Pijn veroorsaeckt, door het geseijde bloed te penetreren: En heb eijntelijck seer naeckt, mij voor de oogen gestelt, in het bloet, (datmen het navel-bloet noemt) ende in het bloet dat ick uijt het Hart nam) dunne pijpjens omtrent twee mael soo langh, als een globule bloet… En dus stelde ick bij mij vast, de oorsaeck vande groote pijn, die het bloet van Ael en Palingh het oogh aenbrengt, ontdeckt te hebben namentl. dat de pijpjens niet alleen de gevoel. deelen van het oogh hebben gequest, maer dat eenige daer in sijn blijven steecken. (Antoni van Leeuwenhoek, Brief No. 33 [21], 5 october 1677).

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SILVERING OF EUROPEAN EEL (Anguilla Anguilla L.): SEASONAL CHANGES OF MORPHOLOGICAL AND METABOLIC PARAMETERS

ABSTRACT

The transformation of yellow eel into silver eel is called ‘silvering’, and takes place prior to migration. We used principal component analysis (PCA) to characterize the morphological and physiological changes that accompany silvering in the European eel (Anguilla anguilla L.). Silvering is positively related to external parameters such as eye size, internal maturation parameters like GSI, vitellogenine (VTG), and blood-substrates like phospholipids, FFA and cholesterol. The Hepatosomatic Index was not significantly different between yellow and silver groups. In contrast, a significant difference was observed for parameters of body constitution (fat, protein, dry-matter) between yellow and silver stages. Furthermore, the process of silvering is accompanied with increased levels of cortisol in fall, which plays a role in mobilization of metabolic energy from body stores towards migratory activity and gonadal growth. Based on PCA analysis with physiological, morphological and endocrinological parameters it is concluded that during the process of 'silvering', several developmental stages can be recognized.

INTRODUCTION

During its life cycle the European eel (Anguilla anguilla L.) experiences two periods of metamorphosis. The first is transformation from the planktonic marine stage (Leptocephalus larvae) into glass eel. This occurs during its oceanic migration from the presumed spawning grounds in the Sargasso Sea to the coasts of Europe before entering fresh water. The second (partial) metamorphosis occurs after the juvenile growth and differentiation phase (> 4 years for males, >7 years for females) in the inland waters. Eels transform then from yellow eel into silver eel, a process called ‘silvering’. During the latter transformation there is some proliferation of the gonads and an increase in eye size (Pankhurst 1982, Pankhurst & Lythgoe 1983). Furthermore, the body colour becomes silvery due to differentiation of pigment cells (Pankhurst & Lythgoe 1982); the alimentary tract shows regression, and the animal becomes fatter. These changes are part of the ‘silvering’ process, which precedes to the spawning migration to the Sargasso, 6000 km away from Europe.

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The mechanisms involved in the onset of ‘silvering’ of eels are largely unknown, as are the different stages, which characterise this metamorphosis. Only two extensive studies have been performed of the morphological and physiological characteristics at the different stages of eel silvering (Durif 2003, Durif et al. 2005). Based on principal component analysis (PCA) she characterized some of the morphological and physiological parameters associated with silvering using the parameters: bodylength, eye index, fin index, condition factor, gonad weight, liver weight, gut weight, gonadotropine and growth hormone (Durif et al. 2005). Seasonal, monthly changes over the year in parameters from the fat metabolism, morphological and physiological parameters have never been described before for female eels from the Grevelingen-lake, a brackish water population. Τhe Grevelingen-lake is the largest brackish/saltwater lake of Western Europe with a total area of 14,000 hectares. The lake is situated on the boundary between Zuid-Holland and Zeeland, The Netherlands and has a large standing stock of eels. This study can be seen as a further refinement of the studies of Durif (2003) and Durif et al. (2005) due to a monthly sampling protocol and taking into account more physiological and metabolic parameters. We hypothesize that the silver eels caught in autumn, which are on their seaward migration, have totally different body characteristics than the sedentary phase caught earlier in spring and summer. Via PCA we will determine which morphological and physiological characteristics are most altered during ‘silvering’. Thus the aim of this study was, by monthly sampling of female European eel at a fixed location (Grevelingen lake, the Netherlands) to describe the transient changes which are characteristic for the process of ‘silvering’, and to determine when these changes first appear. This will help us to understand the dynamics of the transformation process, which is an adaptation to a migration phase in an oceanic environment.

MATERIAL & METHODS

Animals Every month from April until November 2002, eels were caught by local fisherman by fyke nets at the Grevelingen. The 8 largest animals (females) were selected. Water temperature was measured and the eels were classified in ‘yellow’ or ‘silver’ by a fisherman according to external features. These were an enlargement of the eyes and a silvery body

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color in case of the ‘silver’ stage. The fish were rapidly anaesthetized with benzocaine (100 ppm). Blood was collected with a heparinized syringe and stored on dry ice for further analysis. The carcasses were taken to the laboratory to determine the body weight, eye index (E.I.), the digestive tract index (D.T.I.), hepato-somatic index (H.S.I.) and gonad weight (G.S.I.).

Blood analysis In the freshly collected blood samples, treated with anticoagulant, the haemotocrit was measured directly in 9 μl whole blood sample using a haematocrit micro-centrifuge (Bayer, F.R.G.). Hemoglobin content in 20 μl blood was detected after 3 minutes using the cyan-me- themoglobin method (Boehringer Mannheim, F.R.G.). Blood was directly centrifuged (10,000 rpm for 5 min). The plasma was divided in eppendorf tubes (10, 40, 50, 50, 20, 20, 20, 33, 33, 33 μl for respectively total protein, FFA (Free Fatty Acids), glucose, lactate, cholesterol, triglycerids, phospholipids and sodium, potassium and chloride analysis) and stored at -80oC pending analysis. For the glucose measurements, 50 μl plasma was mixed with 200 μl 6% trichloric acid solution to precipitate plasma proteins and stored at -80oC. Glucose was determi- ned by colorimetric assay (Sigma, St.Louis, U.S.A.). FFA was measured with a commercial test-kit WAKO (NEFA C method, Instruchemie, Hilversum, The Netherlands). Lactic acid was determined with an enzymatic test-combination of Boehringer Mannheim: 139084 for L-lactate. Total Protein, Cholesterol, triglycerids, and phospholipids were measured with Boehringer Mannheim test kits (MPR3 124281, MPR1 CHOD-PAP 1442341, GPO-PAP 701882 and MPR2 691844 respectively). Plasma sodium, potassium and chloride levels were measured by flame photometric and colorimetric procedures (Technicon). For cortisol- and vitellogenine measurements the plasma was divided in Eppendorf tubes (25 μl, 50 μl ) and stored at -80oC pending analysis. Cortisol was measured by radioimmunoassay at Nijmegen University according to the protocol of Balm et al. (1994). VTG was measured by immunoenzymatic assay according to the protocol of Burzawa-Gerard et al. (1991).

Carcass analysis After weighing, the total carcass was cut into pieces of about 3 cm and nearly submerged in water in a glass beaker. The samples were autoclaved at 2 atmospheres at 120o for 4 hours. They were then homogenized with a laboratory disperser and subsequently sampled in triplicate for dry matter, protein and fat analyses. The dry matter content was measured by freeze drying of the sub samples to constant weight. Plate temperature started at -20oC and raised to 27oC after a

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vacuum of 40 Pa was reached. Condensor temperature was -90oC. The protein was measured according to ISO 5983 (1979). For the fat determination, the sub-samples were freeze dried, as described for dry matter, and subsequent extraction of the fat was performed as described in ISO/DIS 6492 (1996).

Environmental factors for temporal relationships (correlations) Water-temperature, salinity and day-length over the period April-November 2002 are depicted in figure 1.

Figure 1: Monthly evolution of temperature (°C), salinity, and light (hours) in the Grevelingen in 2002 at the time of sampling. (April is no. 4 until November no. 11).

Calculations and statistics Fulton’s condition-factor (K) was calculated according to the equation K=100*W*L-3. The eye index was calculated according to the method of Pankhurst (1982) where E.I={[(A+B)2/4*π]/L}*100 where A is the horizontal eye diameter, B is the vertical diameter, and L is the total body length (mm). The Hepato somatic Index (H.S.I.) was calculated according to {[Liver weight]/[Body weight]}* 100%. The Gonado somatic Index (G.S.I.) was calculated according to {[Ovary weight]/[Body weight]}* 100%. Morphological and physiological descriptors were tested for normality (Kolmogorov-Smirnov, Lilliefors probability). Those that differed significantly from the normal distribution (p<5%) were log-transformed. To remove any size effect and since mean length of eels slightly differed

54 Chapter 3 between samples, variables which were correlated to length of eels were standardized according to: Var_std=Var-(M(L-L_mean)) (MacCrimmon and Claytor 1986). Where Var_std is the corrected variable, Var is the original variable, M is the slope of the regression of the descriptor on total body length, and L_mean is the mean length of the eels in the sample Means of variables were compared between the yellow eel group and the silver eel group using a two-sample t-test. The variables which did not follow a normal distribution regardless of the log- transformation were compared by Kruskal-Wallis tests. Pearson correlations were calculated between variables, their significance was determined with a Bonferroni test. P≤ 0.05 was considered as statistically significant for all tests. Statistics were performed via Systat SPSS Version 10. Principal Component Analysis (PCA) was carried out on 21 physiological parameters (transformed variables) which would presumably evolve with silvering to examine their seasonal evolution. The PCA was performed with ADE4 (Thioulouse et al. 1997).

RESULTS

The animals caught in the period from April until July were all yellow while the animals in the period September until November were all silver. The month of August comprises both yellow (n=4) and silver (n=3) animals while one animal was classified according to external parameters (skin color, eye size) as ‘half’ silver. In addition, for all measured parameters, an one-way ANOVA was performed on the average value of the yellow vs. the average value for silver animals. In this comparison the ‘half’ silver animal caught in the month of August was eliminated. The largest animals (silver) above 1 kg were found in the period September-November (Table 1). The GSI and eye indexes are the highest in the silver eels (Table 1).

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Principal Component Analysis (PCA) was carried out on a total of 21 physiological variables. The first two axes account for 44% of total inertia. Relationships between factorial axes and variables are indicated on the correlation circle for body constitution and blood substrates (Figure 2, left panel). The first axis accounts for most of the variation (31%); it is significantly correlated to GW and VTG, which are the parameters that reflect the silvering process. Other main contributors to this axis are the following parameters: carcass proteins for negative scores, and carcass fat, dry mass, FFA, triglycerids, phospholipids, cholesterol, for positive scores. Main contributors to the second axis (13% of total inertia) are glucose, carcass-fat, and dry mass for negative scores; cortisol, carcass proteins, and sodium for positive scores.

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April May June July August September October November Yellow Silver P-value Morphology N=8 N=8 N=8 N=8 N=8 N=8 N=8 N=8 N=36 N=28 Body Weight (g) 861±194 969±187 908±187 751±175 771±184 1248±302 1092±132 1178±293 855 ± 195 1132 ± 262 P≤0.0001** Length (cm) 74.9±5.95 77.1±4.76 75.8±5.20 73.4±3.47 70.1±4.90 83.4±5.78 78.4±4.37 79.5±5.81 74.7±5.18 79.2±6.36 P≤0.003** Condition Factor 0.20±0.02 0.21±0.02 0.21±0.01 0.19±0.02 0.22±0.03 0.21±0.02 0.23±0.01 0.23±0.03 0.20±0.02 0.23±0.02 P≤0.0001* Eye-Index 7.34±0.98 7.28±2.38 7.26±0.72 6.69±0.85 7.99±0.93 10.42±0.62 9.95±0.92 10.91±0.86 7.14±1.31 10.17±1.04 P≤0.0001** Gonad Weight (g) 5.75±2.74 6.61±3.31 7.01±2.60 6.19±3.45 7.08±3.83 18.09±5.28 17.02±4.75 16.58±6.25 6.17±2.87 16.16±5.68 P≤0.0001** G.S.I. 0.65±0.15 0.65±0.20 0.76±0.24 0.79±0.28 0.87±0.28 1.44±0.17 1.54±0.30 1.38±0.26 0.71±0.22 1.40±0.28 P≤0.0001** H.S.I. 1.49±0.45 1.28±0.24 1.54±0.50 1.23±0.30 1.17±0.20 1.32±0.09 1.36±0.28 1.37±0.12 1.35±0.39 1.34±0.16 P≤0.924 Digestive Tract (g) 23.9±3.1 33.9±12.4 32.5±16.1 23.4±7.8 13.9±3.8 17.8±4.7 13.4±2.3 13.2±4.5 27.1±11.9 14.8±4.4 P≤0.0001**

Table 1: Mean ± Standard Deviation of morphological parameters that have been studied over their annual cycle of female European eel (Anguilla anguilla L.). From April until July the animals are yellow (non-migratory phase) while from September until November the animals are silver (migratory phase). In August half of the animals is yellow and half of the animals is silver. The mean value of the yellow eel group was compared to the mean value of the silver eel group. Statistics were performed via Systat SPSS using a one-way ANOVA for differences between yellow and silver groups. P≤ 0.05 was considered as statistically significant.

Figure 2: Left panel: Principal Component Analysis (PCA) of body composition and blood substrates; correlation circle. The left panel shows the correlations between the principal components and the variables.

Abbreviations: Carcprot=carcass-protein; SO=Sodium; CL=Chloride; HB=Hemoglobine; CORT=cortisol; TP=total protein; VTG=vitellogenine; CHOL=cholesterol; PL=phospholipids; PO=potassium; TG=triglycerids; FFA=free fatty acids; HCR=hematocrit; GW=gonad weight; K=Fullton's condition factor; LWT=liverweight; CFAT=carcass-fat; DRY= carcass dry matter; LAC=lactic acid; GLUC=glucose; IW=intestine weight.

Right panel: Factorial scores. Individuals (eels) are grouped by month (April (no.4) to November (no. 11). Y: yellow; S: silver. Factorial scores of individuals (eels) are represented on the right panel. The stars each link the eels that were sampled in the same month.

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Table 2: Correlation matrix of the parameters sampled on the eels.

PRO CHO GLU K VTG AW F GW HSI HCR PO PL DRY TCA CL FFA TP LAC CFAT TG HB SO L C RC K 1.00 VTG 0.28 1.00 AW -0.10 -0.53 1.00 F 0.11 0.43 -0.311.00 GW 0.71 0.57 -0.44 0.41 1.00 HSI 0.17 0.01 0.32 0.04 0.10 1.00 HCR -0.03 0.29 -0.130.25 0.14 -0.121.00 PO 0.13 0.13 -0.28 0.11 0.34 0.08 0.00 1.00 CHO 0.28 0.46 -0.36 0.31 0.52 -0.10 0.26 0.19 1.00 L GLU 0.01 -0.10 0.10 -0.14 -0.07 0.23 0.18 -0.10 0.09 1.00 C PL 0.34 0.51 -0.35 0.34 0.58 -0.06 0.33 0.22 0.94 0.05 1.00 DRY 0.58 0.24 -0.25 0.14 0.56 -0.20 0.13 0.24 0.31 0.16 0.40 1.00 PRO TCA -0.58 -0.25 0.08 -0.18 -0.51 0.01 -0.13 -0.15 -0.19 -0.24 -0.29 -0.85 1.00 RC CL 0.02 0.16 -0.02 0.07 0.05 0.08 0.06 0.03 0.07 -0.17 0.14 0.01 0.03 1.00 FFA 0.48 0.31 -0.26 0.24 0.52 -0.07 -0.07 0.14 0.51 -0.20 0.59 0.53 -0.51 -0.02 1.00 TP 0.08 0.11 0.07 0.17 0.18 -0.03 0.15 0.03 0.40 -0.07 0.43 0.06 -0.06 0.09 0.48 1.00 LAC 0.15 0.16 0.00 0.22 0.20 0.00 0.35 -0.11 0.18 0.62 0.20 0.28 -0.32 0.01 -0.19 -0.051.00 CFA 0.59 0.27 -0.24 0.17 0.59 -0.17 0.15 0.25 0.30 0.17 0.40 0.99 -0.88 0.01 0.56 0.05 0.27 1.00 T TG 0.38 0.44 -0.28 0.12 0.57 -0.07 0.20 0.17 0.59 -0.10 0.69 0.47 -0.38 0.07 0.64 0.44 -0.04 0.48 1.00 HB 0.10 0.33 -0.43 0.31 0.27 0.02 0.47 0.03 0.27 -0.04 0.35 0.13 -0.07 0.26 0.13 0.09 0.04 0.13 0.17 1.00 SO -0.09 0.24 -0.290.46 0.07 0.08 -0.02 0.04 0.21 -0.26 0.21 -0.05 0.11 0.45 0.10 0.02 -0.23 -0.04 0.05 0.21 1.00

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April May June July August September October November Yellow Silver P-value Ions & Substrates N=8 N=8 N=8 N=8 N=8 N=8 N=8 N=8 N=36 N=28 Sodium (μM) 345±25 365±36 394±30 422±40 395±48 411±33 388±14 412±44 380 ±44 403 ±31 P≤0.028* Potassium (μM) 3.4±1.1 4.7±1.3 5.6±1.6 4.4±1.5 5.0±2.0 5.2±0.9 6.0±1.5 5.3±1.4 4.4±1.5 5.5±1.5 P≤0.005* Chloride (μM) 328±40 327±40 322±55 335±26 320±33 329±17 315±26 332±38 326±39 326±29 P≤0.992 Total Protein (g/l) 49.7±10.9 49.9±6.6 66.7±18.8 57.1±9.3 52.3±7.4 56.8±6.7 53.8±7.1 49.4±5.5 56.1±13.2 52.4±6.8 P≤0.295 Cholesterol 12.5±4.6 12.1±2.5 14.8±2.0 13.0±3.5 14.2±3.3 17.8±4.2 18.3±7.3 16.5±4.6 13.3±3.2 17.1±5.2 P≤0.001** (mmol/l) Phospholipids 13.1±5.5 13.6±2.6 17.2±2.9 14.1±3.9 15.1±3.6 19.5±2.8 20.4±6.5 19.3±4.9 14.6±3.9 19.2±4.8 P≤0.0001** (mmol/l) Triglycerids 5.6±3.2 5.9±2.6 13.1±6.1 7.7±3.5 9.7±4.8 7.5±1.6 12.2±5.8 11.7±4.3 8.5±5.0 10.4±4.7 P≤0.044* (mmol/l) FFA (mmol/l) 0.17±0.12 0.21±0.14 0.37±0.20 0.24±0.14 0.30±0.11 0.19±0.05 0.24±0.13 0.34±0.12 0.26±0.16 0.26±0.12 P≤0.272 Hb (mmol/l) 7.13±1.40 6.93±1.18 4.84±0.73 7.09±0.87 8.24±2.63 9.11±1.00 7.92±0.56 8.96±0.98 6.75±1.64 8.52±1.51 P≤0.0001** Hct (%) 29.0±7.51 34.7±5.29 33.8±5.30 31.5±4.17 33.5±8.41 49.0±12.1 40.3±3.13 35.8±4.43 32.5±5.65 40.3±9.98 P≤0.0001** Glucose (mmol/l) 1.82±0.43 1.26±0.28 1.52±0.34 0.71±0.18 0.92±0.19 1.43±0.47 1.53±0.21 1.00±0.16 1.28±0.51 1.27±0.38 P≤0.921 Vitellogenin (μg/ml) 104.27 ± 84.03 ± 133.77 ± 159.48 ± 133.84 ± 1023.05 ± 766.81 ± 2438.99 ± 122.01 ± 1189.05 ± P≤0.001** 85.31 8.49 62.45 140.12 34.10 1680.97 1279.3 2626.92 84.53 1883.15 Lactate (mmol/l) 0.70±0.54 0.20±0.12 0.29±0.21 0.10±0.06 0.10±0.04 0.65±0.28 0.76±0.56 0.17±0.08 0.29±0.34 0.45±0.40 P≤0.030* Cortisol (ng/ml) 26.1±10.3 31.5±25.3 27.1±15.5 70.2±38.5 40.7±24.7 108.1±27.7 70.1±55.0 85.8±25.8 38.2±29.1 80.7±42.0 P≤0.0001** Carcass Dry matter (g/kg) 427±41 423±42 428±45 410±38 451±25 450±25 454±17 448±31 424±40 452±23 P≤0.002** Fat (g/kg) 264±52 236±55 244±55 215±48 269±34 269±33 276±21 270±41 235±51 273±31 P≤0.001** Protein (g/kg) 172±11 169±13 168±10 177±38 177±8 165±8 163±5 164±10 171±11 164±8 P≤0.006**

Table 3: Mean ± Standard Deviation of ions, blood-substrates parameters in combination with the steroid cortisol and constitution of the carcass have been studied over their annual cycle of female European eel (Anguilla anguilla L.). From April until July the animals are yellow (non-migratory phase) while from September until November the animals are silver (migratory phase). In August half of the animals is yellow and half of the animals is silver. The mean value of the yellow eel group was compared to the mean value of the silver eel group. Statistics were performed via Systat SPSS using a one-way ANOVA for differences between yellow and silver groups. P≤ 0.05 was considered as statistically significant.

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Table 4: Description of the transformed variables Variable Transformation Abbreviation Body weight Fulton’s condition factor K Gonad weight Log-transformed, GW standardized Intestine weight Log-transformed IW Liver weight % of total body weight HIS Vitellogenin Log-transformed VTG Cortisol Log-transformed F Hematocrit None HCR Hemoglobin None HB Sodium None SO Potassium None PO Chloride Log-transformed CL Cholesterol None CHO Glucose None GLUC Phospholipids None PL Free fatty acids Log-transformed FFA Lactate Log-transformed LAC Triglycerids Log-transformed TG Total protein Log-transformed TP Carcass fat Log-transformed CFAT Carcass protein None PROTCARC Dry matter None DRY

All of these parameters have a seasonal evolution, which coincides with the silvering of eels (Figure 2, right panel). From May to June, eels show no sign of metamorphosis: they still have a low GSI and a high DTI. The levels of glucose and lactate are relatively high. The eels sampled in July are slightly isolated on the factorial plane as they display relatively high values for carcass proteins, cortisol, and sodium compared to previous months. This may reflect the very beginning of the metamorphosis. This sample is also particular, as sodium, chloride and hemoglobin have increased, and values for carcass proteins are at their maximum. On the contrary glucose values are at their minimum.

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The August sample is situated at the very center of the factorial plane, meaning it is composed of eels having intermediate characteristics between the yellow and silver stages. Some of these individuals present signs of metamorphosis, such as a degeneration of the digestive tract as attested by the decrease in IW, and therefore indicating they have stopped feeding. However, GW and VTG concentrations are still very low. It is only during the following period (September to November) that eels show distinctive silver eels features. The low summer GSI of females increases from September until peak migration time in fall. The alimentary tract shows during the period of silvering regression to approximately 1/3 of the values found in spring and summer (table 1). Increased plasma levels of VTG in fall suggest that excess precursor (VTG) was available for producing yolk, the main chemical component of oocyte and ovarian growth: the vitellogenesis process. The correlation coefficient between vitellogenine and GW is statistically significant: 0.58 (p<5%). This implies that the vitellogenine concentration in the blood is a good estimator for gonad growth (GSI). Several other parameters are significantly correlated to GW: cholesterol, triglycerids, phospholipids, dry mass, carcass proteins, condition factor, FFA, and carcass fat. By September and November, plasma cortisol levels are at their highest values during the season just prior to the migration period. It is likely that mobilisation of energy reserves is greatest during this period. Both cholesterol and phospholipids are low until August, and high from September to November, peaking in October. Triglycerids show the same tendency but remain low in September. Although FFA displays some fluctuations, they tend to increase in fall. Haemoglobin continues to increase from August to November. Carcass fat is low during the first part of the year, and high during the second; Carcass protein shows the reverse pattern. Carcass dry matter is low during the first part of the year, and peaks in October. In table 1, the morphological parameters and cortisol have been tested for significant differences between yellow (May to mid August) and silver (half August-November) animals while this has been performed in table 3 for blood substrates and body-carcass constitution. Significant differences (P ≤ 0.05) were observed for potassium, cholesterol, triglycerids, lactic acid, vitellogenin, dry matter, carcass-fat and carcass-protein while highly significant differences (P ≤ 0.0001) were observed for body-weight, eye-index, G.S.I., Digestive Tract, cortisol, phospholipids, Hb and Hct.

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Many significant correlations were found between descriptors (Table 2 & 4). GW was positively correlated with OODIAM (R=0.69), VTG (R=0.57), CHOL (R=0.52), phospholipids (PL, R=0.58), triglycerids (TG, R=0.57). Fat content (CFAT) was significantly related to condition factor (K, R=0.59) and free fatty acids (FFA, R=0.56). Moreover, the eels with the highest fat stores displayed the greatest ovarian development, as all the descriptors indicative of the animals’ energy stores were also correlated with GW (Table 2). PL, FFA, CHO, TG, CFAT were all significantly correlated, with OODIAM as well (Table 2). Lactic acid (LAC) and glucose (GLU) were only significantly correlated with each other (R=0.62).

DISCUSSION

The aim of the PCA analysis was to describe the transient changes, which are characteristic for the process of silvering. Results of the PCA analysis indicate that silvering is positively related by external parameters like eye size, internal maturation parameters like VTG and blood-substrates like phospholipids, triglycerids, FFA and cholesterol. Silvering is also positively related with constituents of the carcass such as protein content and parameters for fuel mobilization like cortisol. In contrast, the PCA analysis indicated that silvering is negatively correlated with intestine weight. Principal Component Analysis (PCA) was carried out on a total of 21 physiological parameters. The first two axes amounted to 44% of total inertia (1st axis 31% and 2nd 13%). So 1/3 of the variation can be explained by one axis, and two explains nearly 50% (44%). This is not extremely much for a PCA but it can be remarked that this manuscript concentrates on metabolic parameters and cortisol while in another manuscript (van Ginneken et al. 2006) important maturation parameters like hormones (oestradiol, testosterone, thyroid hormones and growth hormone), together with oocytediameter, and gonad weight are presented. These parameters accounted for 57% of the total inertia (respectively 42 and 15% for axis 1 and 2). Pankhurst (1982) indicated that the eye index (eye size relative to length) might be used to define an increased stage of ‘silvering’. The cut-off point that separated immature and mature eels was an Eye-index of 6.5. In our study the Eye-index was somewhat higher varying from around 7 in yellow animals towards 10 in silver animals. An intermediate value of 8 was found in August, exactly when the migration season started; half of the animals at this time were yellow and half were silver.

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The differences between the absolute values of Pankhurst (1982) and our values can probably be ascribed to an observer effect. The H.S.I. is more or less constant during the whole year and does not coincide with the levels of VTG and the weight of the alimentary tract regarding both very low correlation coefficients ( r = 0.01 and r = 0.32 respectively). Thus the H.S.I. is not determined by the vitellogenine process or by the supply of food. The H.S.I. is also not significantly different between yellow or silver eel stage (P ≤ 0.924). This result corresponds with the findings of Han et al. (2003) for which they observed that the mean H.S.I. remained constant during silvering for Japanese eel. However, this observation is in contrast to other studies where increased H.S.I. values in silver animals have been reported for European eel (A.anguilla) (Olivereau and Olivereau 1979), and shortfin (A.australis) and longfin (A.dieffenbachii) New Zealand eel (Lokman 1998). The increased content of blood lipids (phospholipids, cholesterol) at silvering in combination with a higher fat content of the body might be an adjustment of the silver eel to its new stage of life. This increased cholesterol level is consistent with studies in maturing salmonids, where increased levels of cholesterol in plasma and gonads during the spawning season were reported (Idler & Tsuyuki 1958, Idler & Bitners 1960). According to Lewander et al. (1974), a redistribution of cholesterol occurs from other tissues to the gonads in silver eel. A low correlation coefficient of (r = -0.24) between the weight of the alimentary tract and the carcass fat content gives does not support the view that the fat composition of the animals is directly dependent on the feeding season and that the fat stores are probably more important for the animals as storage form than nutritional deposition in the liver.

Mesenteric fat has been shown to be important for the hormonal regulation of maturation in Atlantic salmon Salmo salar L. (Rowe et al. 1991) and it has also been suggested that fat content regulates the onset of maturation in eel (Larsson et al. 1990). The assumption that fat is a controlling factor in the initial maturation process is supported by a study of Andersson et al. (1991). In the comparison of yellow Baltic eels vs. Kattegat eels, the individuals originating from the Baltic metamorphose at a greater age. This observation was explained due to the slower accumulation of fat in Baltic yellow eels (Andersson et al. 1991). In our study, fat content of the carcass was very significantly higher in silver animals in comparison with yellow animals (P<0.001).

Many diverse roles have been suggested for cortisol during such metabolic stresses as starvation, osmoregulation, mobilization of energy stores for migration, gonad maturation, spawning (Wingfield & Grimm 1977) and during stress itself (Wendelaar Bonga 1997).

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Cortisol is released from the interrenal tissue when an animal is under exposure to stressors (Wendelaar Bonga, 1997), but changes in cortisol levels can also be attributed to a daily rhythm (Spieler, 1979) and to sexual maturity and season (Pickering and Christie, 1981). Although the stress of capturing the eels with fyke nets, holding them in storage tanks on the boat, and sampling the eels, causes plasma cortisol to rise, we can clearly see an seasonal pattern in cortisol plasma levels. Those changes do indicate a changing activity of the interrenal system throughout the year.

If we compare the pattern of secondary stress parameters of eel like increased glucose, potassium or lactic acid (van Ginneken et al 2002) with the pattern of the cortisol response we find low correlation coefficient of respectively (r=--0.14, 0.11 and 0.22). These low values suggest that this cortisol is not solely involved in a stress-response (Wendelaar Bonga, 1997). At this moment it is not understood whether sexual maturation is accompanied with increased corticosteroid levels (review Idler & Truscott 1972, Pickering 1989). Several studies have demonstrated a correlation between sex hormones, body constitution and cortisol (Mackinnon 1972, Wingfield & Grimm 1977). In general, vertebrates exposed to stress commonly show a reduction of reproductive performance (Moberg 1985). Furthermore, exposure of fish to stress results in impaired reproductive capacity (Donaldson 1990, Barton & Iwama 1991). However in other studies, seasonal elevations in plasma corticosteroids have been found suggesting a positive stimulating role of cortisol in pre-spawning period. Not only in salmonid species like Pacific salmon (Pickering & Pottinger 1987) which only spawn once but also in landlocked non-migratory salmonids which spawn several times like the rainbow trout (Robertson et al. 1961).

In the study reported here, we found elevated cortisol levels in silver eel prior to migration and a rather high correlation with the salinity level (r=-0.79). A role of cortisol in the maturation process of eels is at this moment under investigation at our laboratories. In this study we will simulate the journey of 5500 km to the Sargasso Sea in the laboratory in large Blazka swim-tunnels (unpublished results). However, from our observations in this study it becomes clear that a role for cortisol may be in a mobilization of energy stores. Especially in European eel which have to cover a distance of 6000 km to its spawning areas in the Sargasso. Van Ginneken & van den Thillart (2000) demonstrated in large Blazka swimtunnels of 127 liter that for this tremendous swim effort 40% of the energy reserves are needed while still 60% of their energy stores can be used for gonad development.

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The observation that blood-substrates like phospholipids and cholesterol are significantly increased in European silver eels corroborate the view that the major role of cortisol lays in the mobilization of the energy stores prior to migration.

Based on PCA analysis with physiological, morphological and endocrinological parameters we see in figure 2B that during the process of 'silvering', several developmental stages can be recognized. Up to now silvering was split into two separate stages: yellow and silver. This classification did not take into account a possible preparatory phase. Feunteun et al. (2000) classified eels into three stages: yellow, silver and yellow/silver.

However, these stages were only based on external and visual variables (skin color, visibility of the lateral line and eye surface). This is in our opinion the first study were physiological parameters (blood substrates, constitution carcass) together with endocrinological parameters (cortisol) were incorporated in the analysis giving a finer seasonal description of the silvering process (start and duration) as well as of the physiological mechanisms involved (triggers and endocrine control).

ACKNOWLEDGEMENTS

We thank the fishermen Wim and Piet Bout (Brunisse, The Netherlands) for supplying Grevelingen eels every month. This study was supported by the Netherlands Organisation for Scientific Research (STW-project no. LBI66.4199) and by the European Commission (project QLRT-2000-01836). Prof.Dr.S. Dufour and Dr. M. Sbaihi (Museum Mational Histoire Naturelle, Paris) are kindly acknowledged for providing vitellogenin data. Prof.M.Richardson is acknowledged for helpful suggestions and improvement of the English of the manuscript.

LITERATURE

Andersson, J., Sandström, O. and Hansen, H.J.M.(1991). Elver (Anguilla anguilla L.) stockings in a Swedish thermal effluent –recaptures, growth and body conditions. J.Appl.Ichthyol. 7, 78-89. Balm, P.H.M., Pepels, P., Helfrich, S., Hovens, M.L.L. & Wendelaar Bonga, S.E. (1994).Adrenocorticotropic hormone (ACTH) in relation to interrenal function during stress in tilapia (Oreochromis mossambicus). General Comparative Endocrinology 96, 447-460.

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Barton, B.A. & Iwama, G.K.(1991). Physiological changes in fish from stress in aquaculture with emphasis on the responses and effects of corticosteroids. Annu.Rev.Fish.Dis. 1, 3- 26. Burzawa-Gérard, E.' Nath, P., Baloche, S., Peyon, P. (1991). ELISA (enzyme-linked -immunosorbent assay) for vitellogenin and vitellus in the eel (Anguilla anguilla) and in the Indian major carp (Labeo rohita). In: Scott AP, Sumpter JP, Kime DE Rolfe MS (eds.), Reproductive Physiology of Fish. Sheffield: Fish Symp: 1991, 319 Donaldson, E.M.(1990). Reproductive indices as measures of the effects of environmental stressors in fish. Am.Fish.Soc.Symp. 8, 109-122. Durif, C. (2003). The downstream migration of the European eel Anguilla anguilla: Characterization of migrating silver eels, migration phenomenon, and obstacle avoidance. PhD Thesis, University Paul Sabatier, Toulouse. Durif, C.; Dufour, S.; Elie, P. (2005). The silvering process of Anguilla anguilla: a new classification from the yellow resident to the silver migrating stage. J.Fish.Biol. 66, 1025-1043. Feunteun, E.; Acou, A., Lafaille, P & Legault, A.(2000). The European eel: prediction of spawner escapement from continental population parameters. Canadian Journal of Fisheries and Aquatic Sciences 57, 561-570. van Ginneken, V.J.T. &. van den Thillart, G.E.E.J.M (2000). Eel fat stores are enough to reach the Sargasso. Nature 403, 156-157. van Ginneken, V.J.T.; Balm, P., Sommandas, V., Onderwater, M., Van den Thillart, G. (2002). Acute stress syndrome of the yellow European Eel (Anguilla anguilla Linnaeus) when exposed to a graded swimming load. Neth.J.Zoology 52, 29-42. Van Ginneken, V. ; Durif, C. ; Dufour, S. ; Sbaihi, M. ; Boot, R. ; Noorlander, K. ; Doornbos, J. ; Murk, A.J. ; G.van den Thillart (2006). Endocrine and metabolic profiles during silvering of the European eel (Anguilla anguilla L.). Comp.Biochem.Physiol. submitted. Han, Y-S., Liao, I.-C., Tzeng, W-N.; Huang, Y-S., Yu, J Y-L. (2003). Serum estradiol-17β and testosterone levels during silvering in wild Japanese eel Anguilla japonica. Comp.Biochem.Physiol. B 136, 913-920. Idler, D.R. & Tsuyuki, H.(1958). Biochemical studies on sockeye salmon during spawning migration. 1: Physical measurements, plasma cholesterol and electrolyte levels. Can.J.Biochem.Physiol. 36, 783-791.

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Idler, D.R. & Bitners, I. (1960). Biochemical studies on sockeye salmon during spawning migration. IX: Fat, protein and water in the major internal organs and cholesterol in the liver and gonads of the standard fish. J.Fish.Res.Bd.Can. 17, 113-122. Idler, D.R. and Truscott, B.(1972). Corticosteroids in fish. In: Steroids in Non- mammalian Vertebrates (Edited by Idler D.R.) pp. 127-252. Academic Press, New York. ISO 5983, (1979). (International Organisation for Standardisation), Animal feeding stuffs- Determination of nitrogen content and calculation of crude protein content, 1979 (ISO 5983), Geneva, Switzerland. ISO/DIS 6492, (1996). (International Organisation for Standardisation), Animal feeding stuffs- Determination of nitrogen content and calculation of fat content, category B, 1996 (ISO/DIS 6492), Geneva, Switzerland. Larsson, P., Hamrin, S. & Okla, L.(1990). Fat content as a factor inducing migratory behaviour in the eel (Anguilla anguilla L.) to the Sargasso Sea. Naturwissenschaften 77, 488-490. Lewander, K., Dave, G., Johansson, M.L., Larsson, A. & Lidman, U. (1974). Metabolic and haematological studies on the yellow and silver phases of the European eel, Anguilla anguilla L. I: Carbohydrate, lipid, protein and inorganic ion metabolism. Comp.Biochem.Physiol. 47B, 571-581. Lokman, P.M.; Vermeulen, G.J.; Lambert, J.G.D. & Young, G.(1998). Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels (Anguilla dieffenbachi and A.australis) before and at the onset of the natural spawning migration. I. Females. Fish Physiology and Biochemistry 19, 325-338. MacCrimmon, H.R. & Claytor, R.R.(1985). Meristic and morphometric identity of Baltic stocks of Atlantic Salmon (Salmo salar). Can.J.Zool. 63, 2032-2037. Mackinnon, J.C.(1972). Summer storage of energy and its use for winter metabolism and gonadal maturation in American plaice (Hippoglossoides platessoides). J.Fish.Res.Bd.Canad. 29, 1749-1759. Moberg, G.P.(1985). Influence of stress on reproduction: measure of well-being. In: Animal Stress, edited by G.P.Moberg, Bethesda, MD. Am.Physiol.Soc., 1985, 27-49. Oliverau, M. & Oliverau, J.(1979). Effect of estradiol-17β on the cytology of the liver, gonads and pituitary, and on plasma electrolyte in the female freshwater eel. Cell.Tiss.Res. 199, 431-454.

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Pankhurst, N.W.(1982). Relation of visual changes to the onset of sexual maturation in the European eel Anguilla anguilla (L.). J.Fish.Biol. 21, 127-140. Pankhurst, N.W. & Lythgoe, J.N. (1982). Structure and colour of the integument of the European eel Anguilla anguilla (L.). J.Fish.Biol. 21, 279-296. Pankhurst, N.W. & J.N. Lythgoe, (1983). Changes in vision and olfaction during sexual maturation in the European eel Anguilla anguilla (L.). J.Fish.Biol. 23, 229-240. Pickering, A.D.& Christie, A.A.(1981). Changes in the concentrations of plasma cortisol and thyroxine during sexual maturation of the hatchery-reared brown trout, Salmo trutta L. Gen.Comp.Endocrinol. 44, 487-496. Pickering, A.D. & Pottinger, T.G.(1987). Lymphocytopenia and interrenal activity during sexual maturation in the brown trout, Salmo trutta L. J.Fish.Biol. 30, 41-50. Pickering, A.D.(1989). Environmental stress and the survival of brown trout, Salmo trutta L.; A review. Fresh Water Biology 21, 47-55. Robertson, O.H.; Krupp, M.A.; Thomas, S.F.; Favour, C.B.; Hane, S. & Wexler, B.C.(1961). Hyperadrenocorticism in spawning migratory and nonmigratory rainbow trout (Salmo gairdnerii) comparison with Pacific salmon (genus Oncorhynchus). Gen.Comp.Endocrinol. 1, 473-484. Rowe, D.K.; Thorpe, J.E.& Shanks, A.M.(1991). Role of fat stores in the maturation of male Atlantic salmon (Salmo salar) parr. Can.J.Fish.Aquat.Sci. 48, 404-413. Spieler, R.E.(1979). Diel rhythms in circulating prolactin, cortisol, thyroxine, and triiodothyronine levels in fishes. A review. Rev.Can.Biol. 38, 301-315. Thioulouse, J., Chessel, D., Dolédec, S. & Olivier, J. M. (1997). ADE-4: a multivariate analysis and graphical display software. Statistics and Computing 7, 75-83. Wendelaar Bonga, S. (1997). The stress response in fish. Physiological Reviews 77, 591- 625. Wingfield, J.C. & Grimm, A.S.(1977). Seasonal changes in plasma cortisol, testosterone and oestradiol-17β in the plaice, Pleuronectes platessa L. General and Comparative

Endocrinology 31, 1-11.

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ENDOCRINE AND METABOLIC PROFILES DURING SILVERING OF THE EUROPEAN EEL (Anguilla anguilla L.).

V. van Ginneken1; C. Durif2; S. Dufour3; M. Sbaihi3; Ron Boot1; K.Noorlander1, J.Doornbos1, A.J.Murk4; G.van den Thillart1.

1) Integrative Zoology, Institute Biology Leiden (IBL), van der Klaauw Laboratorium, P.O.Box 9511, 2300 RA Leiden, The Netherlands. 2) Department of Biology, University of Oslo, P.O. Box Blindern, 0316 Oslo, Institute of Marine Research-Austevoll, 5392 Storebø, Norway 3) Museum National Histoire Naturelle (MNHN), Paris, France. 4) Department Toxicology, Wageningen University, P.O..Box 8000, 6700 EA Wageningen, The Netherlands.

Corresponding Author: Dr.V.J.T.van Ginneken, Integrative Zoology, Institute Biology Leiden (IBL), van der Klaauw Laboratorium, P.O.Box 9511, 2300 RA Leiden, The Netherlands, FAX: +31(0)71-5274900, E-mail: [email protected], TEL: +31(0)71-527749

KEY-WORDS: European eel; (Anguilla anguilla L.); growth hormone, thyroid hormone, cortisol, testosterone, estradiol, seasonal changes, metamorphosis, silvering.

Submitted to: CBP

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Bestudering van de werking van het palinghart Wanneer ik eenige tijd geleden, de Herten van verscheijde Vissen beschouwde, ende wel die vande groote Ael, en paling, soo nam ik vermaak, om deselve veel maal te besien, ende dat, om dat soo danigen Hert, wel vier uren lang in beweginge was, na dat het uijt de Vis was genomen, te meer, om datter soo een ordentelijke beweginge wierde te weeg gebragt, want als het Bloet uijt het Hert wierde gestooten, soo en wierde het met die vaardigheijt, niet gebragt inde groote Arterie, want die soude met die vaardigheijt, de menigte van Bloet, niet konnen als over nemen, ende dus wierde het Bloet, uijt het Hert komende, gestort in een wit peers gewijse lighaamtje, dat men wel voor een blaasje soude aan sien, en welkers eene opening vereenigt was, aan de groote Arterie, ende de andere opening, was vereenigt aan het Hert, en al waar een klap-vlies is, om dat wanneer het Bloet, in dit peers gewijse deel is in gestort, niet weder in het Hert, soude konnen te rugge loopen…………….. Gelijk nu, dit geduijrig voort stooten van het Bloet, inde Arterie, sonder punct des tijts stilstant, soo beelt ik mij in, gaat het soo ordentelijk toe in het voort stooten van het Bloet, uijt het Hert, inde Arterien der Dieren, te meer om dat wij doorgaans gewaar werden, dat de Heere Maker van het Geheel Al, desselfs Uijtwerkinge, inde groote bewegende schepsels, op een ende deselve wijse te weeg brengt, schoon de Herten, van een geheel andere maaksels sijn, als die vande Vissen (Antoni van Leeuwenhoek, Brief No. 277, 28 augustus 1708).

Chapter 4

ENDOCRINE AND METABOLIC PROFILES DURING SILVERING OF THE EUROPEAN EEL (Anguilla anguilla L.)

ABSTRACT

The transformation of yellow eel into silver eel is called ‘silvering’, and takes place prior to migration. This is the first study to provide hormonal profiles of European eel (Anguilla anguilla L.) during the silvering. This transformation occurs in association with hormonal surges of testosterone (T) and estradion (E2) but not with these of thyroid hormones (TH) and growth hormone (GH) which have a maximum activity in spring and a minimum activity in summer and autumn. It is therefore suggested TH’s and GH are not important for eel gonadal development in the autumn. Based on PCA analysis with physiological, morphological and endocrinological parameters it is concluded that the transition is gradual and that eels go through several stages.

INTRODUCTION The European eel (Anguilla anguilla L.) undergoes two metamorphoses during its life cycle. The first one corresponds to the transformation from leptocephalus larvae into glass eel during its oceanic migration from the supposed spawning grounds in the Sargasso Sea to the European coasts. The second metamorphosis occurs after the growth phase as a yellow eel, and marks the onset of puberty (silver eel). This transition phase, referred to as silvering, anticipates the long-distance migration back to oceanic waters. The drastic changes of habitat (from a freshwater or coastal habitat to an open-sea environment) and behavior (from sedentary to migratory) necessitate the modification of many systems (see Lokman et al. 2003 for review): visual (increase in eye-surface, and shift to shorter wave-lengths characteristic of deep-sea vision, Archer et al. 1995), osmoregulatory (hypo-osmoregulatory adaption), and metabolic (increase in muscle power output, and changes in location of fat stores). Silver or migratory eels have stopped feeding, and a regression of the alimentary tract can be observed. The most important changes are related to the reproductive system. Although true sexual maturation only occurs during the oceanic migration, the gonadotropic axis is initiated before the eel starts its first downstream movements. A slight development of gonads can be observed in female silver European eels while they are still in fresh- or coastal waters.

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Unlike smoltification in Salmonids, silvering of eels is largely unpredictable. It occurs at various ages (5-20 years) and sizes (body length: 26-101 cm) (Tesch 2003; Dekker et al. 1998). Because of the difficulty of getting individuals while they are in the process of metamorphosing, it is most common when studying eels, to separate individuals into two groups, yellow (resident) and silver (presumably migrant), and to compare the physiological profiles between the two. Basic knowledge was obtained in this way on the major differences in hormone levels between the two stages. Histology has shown that the thyroid gland of silver eels is more active than that of yellow eels (Callamand and Fontaine 1942). High levels of TT4 have been found in migratory eels (Marchelidon et al. 1999). The production of thyroid hormones is also thought to be responsible for the hyperactivity of eels at the onset of migration (Fontaine 1975). The most important changes however relate to the reproductive system. As in all vertebrates, the development and activity of the gonads are under the positive control of gonadotropic hormones (GTH), which are produced by the pituitary. In response to stimulation, gonads will produce gametes and sexual steroids, which in turn will stimulate other organs implicated in reproduction. Vitellogenin is secreted by the liver under the control of estradiol. In contrast to yellow eels, silver eels only, have cells with the necessary estradiol receptors (Burzawa-Gerard et al. 1994). Yellow eels are incapable of vitellogenesis, while silver eels also have the structures, which will allow endocytosis of vitellogenin. In the yellow eel, the gonadotropic function is totally inactive, while in silver eels plasma levels of gonadotropin indicate a weak activity (Dufour et al. 1983a, Dufour et al. 1983b). Although main differences between the two stages have been described, little is known about the dynamics of the silvering process, although it has been shown that the transition is gradual and that eels go through several stages (Durif et al. 2005). The objective of this study was to obtain a finer seasonal description of the silvering process (start and duration) as well as of the physiological mechanisms involved (triggers and endocrine control). Due to the difficulty to obtain early silvering eels or to predict eels that will start the metamorphosis, a monthly sampling schedule was carried out in which the biggest eels were collected as they were the most likely to start silvering. Eels were sampled at regular intervals from April to November, and several endocrine and metabolic parameters were analyzed.

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MATERIAL & METHODS

Animals Every month from April until November 2002, eels were caught by local fishermen by fyke nets at the Grevelingen. This is the largest saltwater lake of Western Europe with a total area of 14,000 hectares. The lake is situated on the boundary between Zuid-Holland and Zeeland, The Netherlands. As they were most likely to start silvering, the largest 8 female eels were selected every month (body length 63-90 cm). The fish were rapidly anaesthetized with benzocaine (100 ppm). Blood was collected with a heparinized syringe and stored on dry ice for further analysis. The carcasses were taken to the laboratory to measure body length, body weight, vertical and horizontal eye diameters. Organs were collected to determine the weights of digestive tract, liver, and gonads.

Blood Analysis: For hormones and vitellogenin (VTG) the plasma was divided in Eppendorf tubes (25 μl, 50 μl, 100 μl, 1 ml, 1 ml) for measurement of VTG, growth hormone (GH) testosterone (T) and 17-β-estradiol (E2), and stored at -80oC pending analysis. VTG was measured by immunoenzymatic assay according to the protocol of Burzawa-Gerard et al. (1991) and GH by radioimmunoassay according to Marchelidon et al. (1996). T and E2 measurements were performed by Radio-Immuno-Assays. Total thyroxin (TT4), free-T4 and tri-iodothyronine (TT3) were determined with commercial Amerlite kits (Amsersham International PLC, UK) modified for fish plasma.

Histology After fixation in Bouin’s fluid gonads were dehydrated in a graded ethanol series and embedded in Historesin according to standard procedures (Romeis 1968). They were sectioned at 5 μm and stained with haematoxylin and eosin. The length and width of thirty oocytes of one section were measured and then averaged.

Data analysis Morphological and physiological descriptors were tested for normality (Kolmogorov-Smirnov, Lilliefors probability). Those which differed significantly from the normal distribution (p<5%)

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were log-transformed (gonad weight, alimentary tract weight, FT4, TT4, TT3, oocyte diameter, testosterone, VTG, and GH). Hepato-somatic index (HSI) was calculated as =liver weight/body weight X100. Other common indices, such as gonado-somatic index for gonad weight, were not used as they were correlated to body length of eels and mean length of eels differed between monthly samples. Therefore, to remove any size effect, variables which were correlated to length of eels were standardized according to: Var_std=Var-(M(L-L_mean)) (MacCrimmon and Claytor 1986). Where Var_std is the corrected variable, Var is the original variable, M is the slope of the regression of the descriptor on total body length, and L_mean is the mean length of the eels in the sample. Three variables were standardized in this way: log-transformed gonad weight, liver weight, and log-transformed testosterone. Table 1 summarizes the different variables and transformations. Pearson correlations were calculated between the transformed variables, and their significance was determined with a Bonferroni test. P≤ 0.05 was considered statistically significant for all tests. Statistics were performed via Systat SPSS Version 10. Principal Component Analysis (PCA) was applied to examine the simultaneous variations of hormone levels and to detect common physiological profiles between individuals. Three internal morphological parameters were added as they are known to vary significantly during silvering (gonad weight, oocyte diameter, and alimentary tract weight). The analysis was carried out on the following standardized variables: E2, VTG, GW, TEST, TT4, TT3, OODIAM GW, and AW (figure 1). The PCA was performed with ADE4 (Thioulouse et al. 1997). Missing values were most frequent for GH level, and its contribution to the axes was not significant. Its variations were presented separately (figure 2).

RESULTS The first two axes of the Principal Component Analysis (PCA) accounted for 57% of the total inertia (respectively 42 and 15 % for axes 1 and 2). Axis 1 was positively correlated with variations in alimentary tract weight (AW), and negatively correlated with estradiol (E2), testosterone (T), gonad weight (GW), vitellogenin (VTG) and oocyte diameter (OODIAM) (Figure 1, left panel). Axis 2 represented variations in tri-iodothyronine and total thyroxin (TT3 and TT4). Individuals formed tight clusters on this factorial plot, which corresponded to each monthly sample (Figure 1, right panel).

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Table 1: Description of the transformed variables Variable Transformation Abbreviation Body weight Fulton’s condition factor K Gonad weight Log-transformed, GW standardized Alimentary tract weight Log-transformed AW Liver weight % of total body weight HSI Oocyte diameter Log-transformed OODIAM Estradiol None E2 Testosterone Log-transformed, T standardized Vitellogenin Log-transformed VTG Free thyroxin Log-transformed FT4 Total thyroxin Log-transformed TT4 Tri-iodothyronine Log-transformed TT3 Growth hormone Log-transformed GH

Figure 1: Seasonal evolution of physiological characteristics of eels sampled between April and November: testosterone (T), estradiol (E2), vitellogenin (VTG), total thyroxin (TT4), tri- iodothyronine (TT3), gonad weight (GW), alimentary tract weight (AW), and oocyte diameter (OODIAM). Left panel: Correlation circle of variables. Right panel: Factorial scores of individuals (eels). Clusters correspond to the months that were sampled (April to November).

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Therefore eels caught during the same month displayed very similar physiological profiles. Monthly samples of eels were regularly distributed along the first axis indicating a seasonal evolution in which four major phases could be described. The first phase corresponded to the month of April, during which eels exhibited high levels of TT3 and TT4 (up to 29.4 and 75.4 nmol/l respectively). Samples from May and June were quite similar and displayed much lower levels of TT3 and TT4. They were mainly characterized by high AW, indicative of active feeding. The transition phase (to silvering individuals) seemed to occur during July and August, as noted by the regression of the alimentary tract (decrease of AW). While levels of TT3 and TT4 further decreased in July, they showed a slight increase in August. The last phase (September to November) corresponded to an overall increase of the parameters linked to the onset of puberty (E2, T, VTG, OODIAM). E2 stabilized between September and November, while VTG and OODIAM increased progressively during this period. TT3 and TT4 levels decreased again in November. Levels of GH showed high within month variability especially between April and June (Figure 2). Certain individuals reached very high values (maximum of 0.325 μg/g in June, while the minimum was 0.015 (μg /g in May). Mean and standard deviation decreased there onwards (starting at the transition phase (July and August). Levels remained low until November. Histology showed that the ovaries of eels caught in April and August were characterized by previtellogenic oocytes, those of the November eels were dominated by oocytes in the lipid vesicle stage (Figure 3). 0.4 Figure 2: Monthly evolution of growth hormone (GH in μg/g) from April until November. 0.3

g/g) Mean ± SD (n = 6-8 individuals). μ

0.2 Growth Hormone ( Hormone Growth 0.1

0 4 5 6 7 8 9 10 11 Month

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Figure 3:Example of the annual development of the oocytes of eels. Per month the 8 largest caught female animals were selected. The oocytes in april are in the previtellogenic or early vitellogenic stage containing peripheral yolk granules. The oocytes of the month August and November are in a more advanced stage called the lipid vesicle stage or cortical alveolair stage, which can be characterized by oocytes with oil droplets.

DISCUSSION

Energy stores and growth hormone This study is the first to provide hormonal profiles of European eels during the silvering process. Sampling was not carried out randomly, as the 8 largest females were chosen. This was to ensure that some of the sampled individuals would be undergoing silvering. Surprisingly, there was little within-month variability in their hormonal profiles, indicating that the eels collected on the same month were all undergoing similar endocrine changes. This suggests that starting at a certain size (here 60 cm), eels may all start the first steps of the silvering process. Whether they all go through the final phases of the metamorphosis and actually start their spawning migration may be determined later and depend on the eel’s fat stores. Growth hormone (GH) is firstly implicated in body growth and development of organs and tissues in young animals. However, it has many roles in other physiological processes among which reproduction and osmoregulation by stimulating the production of thyroid hormones (Evans 1993). For salmonids it is suggested that GH and TH’s both may play an osmoregulatory role (Sakamoto et al. 1993). The close link to each other has been observed in one molecular study were it has been demonstrated that regulation of the growth hormone gene in salmon was mediated by among all thyroid receptors (Sternberg & Moav 1999). In salmonids elevated GH is associated with developmental and osmoregulatory changes during processes like smoltification, migration and entry into seawater (Sakamoto et al. 1993).

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For Salmonids it is reported that decreasing temperatures may be the trigger for smoltification (Boeuf 1994). For eel however, GH probably has no role in osmoregulation because hypophysectomized eel can survive in both fresh or seawater (Oliverau & Ball 1970), while hypophysectomized salmonids only survive in freshwater (review: Sakamoto et al. 1993). The action of GH is mediated by insulin-like growth factor I (IGF-I). Messaouri-Deboun et al. (1991) showed that GH has a stimulatory effect on estradiol receptors in the liver. These receptors are then able to induce vitellogenesis after fixation of the ligand. GH controls the release of IGF-I (produced by the liver). Huang et al. (1998) have studied its influence on the activation of puberty in eel. IGF-I exerts a negative feedback on the animal’s growth by inhibiting GH release at the pituitary level and by activating GtH production and therefore steroid synthesis. Estradiol and testosterone would in turn stimulate GtH by inhibiting GH. IGF-I would act as a switch between somatic growth and the onset of puberty. Our results support this, as GH levels in spring were higher than in autumn, as they decreased during the transition phase (July-August). GH values in spring showed high variability. This may have reflected the fact that some of these eels (with low GH levels) would not have completed their metamorphosis, and would have waited another season.

Thyroid hormones Swift (1960) states in his review that, in principle, there appear two types of activity of the thyroid gland of fish living in our latitudes. One type is connected with gonad maturation and results in an increased activity of the gland at spawning time. The second type of activity occurs in a regular annual cycle in both mature and immature fish (Swift 1960). Other studies on eel also showed increased thyroid and pituitary activity during silvering (Callamand and Fontaine 1942, Etienne 1959, Knowles & Vollrath 1966). Also in the study of Han et al. (2004), serum thyroxine levels increased in parallel with TSH β mRNA expression during silvering, supporting the hypothesis that the hypothalamus-pituitary-thyroid axis is correlated to silvering in the wild Japanese eels. However results from this study emphasise the importance of the annual cycle for thyroid hormones and its interaction with other hormones. The peak in TT3 and TT4 observed in our study in April can be indicative for an increased thyroid activity at the onset of the silvering process. In general, for many fish species living in our latitudes, there is a maximum activity of the thyroid gland during the winter and spring, and minimum activity during the summer.

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This cycle has been observed in the trout, the minnow, cod and Fundulus heteroclitus (review: Swift 1960). In the climbing perch, Anabas testudineus, it was observed that TT4 reaches its maximal concentration in spring at the beginning of the spawning season (Chakraborti and Bhattacharya 1984). Also in salmons during parr-smolt transformation thyroxin is involved and a peak was found in salmon plasma in April (Dickhoff et al. 1978). In principle, there are two environmental factors that vary with the same rhythmical fashion each year: the daily photoperiod and the water temperature (Lam 1959). Swift (1960) suggests that the cycle of activity results from the combination of both factors: water temperature and daily photoperiod, temperature being the major controlling factor.

Steroids

Until now it is assumed androgens like T appear to play a more indirect role in reproduction of female fish. They are produced in response to gonadotropin by the thecal layers of the oocytes and the levels gradually increase during the oocyte growth and peaks during postvitellogenic growth (Nagahama 1994). They have in principle a triple function. First, from the well-marked seasonal pattern in female T, which lags behind but follows female E2, we can conclude there is a causal relationship between the two (R=0.56, Table 2). This close relationship between the two steroids supports the possibility that testosterone may act as a precursor for E2 synthesis during the vitellogenic season via aromatizing activity. The increased E2 profile in the period September-November suggests that in the period of gonad development the aromatizing enzymes are partially stimulated. In several fish species aromatizing activity have been observed in the ovary (review: Nagahama 1994). A second, indirect role of T in reproduction is its role in hypothalamic steroid feedback e.g. in acting to increase pituitary GtH production via stimulation of gonadotropin releasing hormone (Montero et al. 1995). Third, from in vitro studies on oocytes of amago salmon it is suggested that T enhances the rate of Germinal Vesicle Breakdown in response to gonadotropin via the conversion to DHPG (Ueda et al. 1984). However for eels in general T and 11-keto-T may play a more prominent role in females during silvering and/or maturation. Lokman et al. (1998) found high values of 11-Ketotestosterone (11-KT) in females of Anguilla dieffenbachi, and suggested that this steroid may play a role in preparing maturing animals for their spawning migration. Indeed, Rohr et al. (2001), demonstrated in immature short- finned eels (A. australis) implanted with a vehicle containing 11-KT, that this steroid was involved in the process of silvering.

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Observed changes were: a) a change in head shape and pectoral fin appearance, b) structural changes of the skin, and c) an increase in eye size and ventricular, liver and gonad mass (Rohr et al. 2001). Also Han et al. (2003) find that androgen, but not estrogen, plays a major role in silvering process of the eels in both sexes. Although we found a low correlation coefficient of (R=0.31, Table 2) between plasma E2 and VTG it is generally assumed that there is a causal relation between those two components in the vitellogenesis which is at the basis for the growth of the gonad by incorporation of yolk proteins in the oocytes (review: Nagahama 1994). The low correlation coefficient of 0.31 can be explained by the substantial increased levels of VTG in fall, in comparison with the stabilization of plasma E2 from September to November. The progressively larger vitellogenic response can be ascribed the so called ‘memory effect’, an increased sensitivity of the liver to E2 via its receptor mechanism (sensitivity and density) and also possibly by an enhanced post-transcriptional mechanism of hepatic vitellogenesis (Jackson & Sullivan 1995).

Table 2: Correlation matrix of the parameters sampled on the eels. K E2 VTG AW FT4 TT4 TT3 OODIAM GW T H.S.I. K 1.00 E2 0.29 1.00 VTG 0.28 0.31 1.00 AW -0.10 -0.27 -0.53 1.00

FT4 -0.17 -0.12 -0.26 0.09 1.00 TT4 -0.01 0.00 -0.14 -0.07 0.75 1.00 TT3 0.06 0.05 -0.05 0.04 0.25 0.45 1.00 OODIAM 0.33 0.29 0.68 -0.66 -0.33 -0.20 -0.12 1.00 GW 0.71 0.43 0.57 -0.44 -0.24 -0.07 0.02 0.69 1.00 T 0.43 0.56 0.46 -0.39 -0.13 0.02 -0.03 0.32 0.48 1.00 H.S.I. 0.17 0.08 0.01 0.32 -0.06 0.03 -0.07 -0.03 0.10 0.11 1.00

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Table 3: Morphological and endocrine parameters examined over an 8-month period in female European eel (Anguilla anguilla L.). Means ± SD

April May June July August September October November Morphology N=8 N=8 N=8 N=8 N=8 N=8 N=8 N=8 Body Weight (g) 861±194 969±187 908±187 751±175 771±184 1248±302 1092±132 1178±293 Eye-Index 7.34±0.98 7.28±2.38 7.26±0.72 6.69±0.85 7.99±0.93 10.42±0.62 9.95±0.92 10.91±0.86 Gonad Weight 5.75±2.74 6.61±3.31 7.01±2.60 6.19±3.45 7.08±3.83 18.09±5.28 17.02±4.75 16.58±6.25 (g) G.S.I. 0.65±0.15 0.65±0.20 0.76±0.24 0.79±0.28 0.87±0.28 1.44±0.17 1.54±0.30 1.38±0.26 H.S.I. 1.49±0.45 1.28±0.24 1.54±0.50 1.23±0.30 1.17±0.20 1.32±0.09 1.36±0.28 1.37±0.12 Alimentary Tract 23.9±3.1 33.9±12.4 32.5±16.1 23.4±7.8 13.9±3.8 17.8±4.7 13.4±2.3 13.2±4.5 Weight (g) Oocyte Diameter 0.086± 0.014 0.096± 0.011 0.108± 0.123± 0.137± 0.145± 0.005 0.159± 0.173 ± 0.011 (mm) 0.018 0.011 0.013 0.013

Maturation TT4 (nmol/l) 36.5±24.6 16.6±17.1 17.2±27.4 7.4±5.1 13.5±5.4 13.5±5.0 12.0±5.9 7.6±5.4 Free T4 (pmol/l) 114.6±143.9 28.3±24.4 63.3±138.6 17.7±8.7 22.4±8.5 21.8±6.5 18.3±12.4 12.0±6.3 TT3 (nmol/l) 12.4±8.8 6.4±2.8 18.3±11.7 5.5±5.1 10.3±5.7 10.4±5.9 6.8±5.3 6.2±5.0 Growth-hormone 0.131± 0.097 0.0869± 0.155± 0.045± 0.085± 0.055± 0.021 0.083± 0.057 ± 0.026 (μg/g) 0.083 0.011 0.018 0.052 0.050 Oestradiol 1.83±0.31 1.64±0.69 1.11±0.60 0.97±0.37 1.83±0.75 2.43±0.60 2.49±0.42 2.09±0.63 (ng/ml) Testosteron 0.75±0.31 0.56±0.25 0.47±0.17 0.42±0.22 0.41±0.15 1.04±0.25 1.04±0.52 1.69±1.27 (ng/ml) Vitellogenin 104.27± 84.03± 133.77± 159.48± 133.84± 1023.05± 766.81± 2438.99± (μg/ml) 85.31 8.49 62.45 140.12 34.10 1680.97 1279.3 2626.92

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ACKNOWLEDGEMENTS We thank the fishermen Wim and Piet Bout (Brunisse, The Netherlands) for supplying Grevelingen eels every month. This study was supported by the Netherlands Organisation for Scientific Research (STW-project no. LBI66.4199) and by the European Commission (EELREP,-Q5RS-2001-01836). Prof. M. Richardson is acknowledged for helpful suggestions and improvement of the English of the manuscript.

LITERATURE CITED

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Burzawa-Gérard, E., Nath, P., Baloche, S., Peyon, P., 1991. ELISA (enzyme-linked- immunosorbent assay) for vitellogenin and vitellus in the eel (Anguilla anguilla) and in the Indian major carp (Labeo rohita). In: Scott AP, Sumpter JP, Kime DE Rolfe MS (eds.), Reproductive Physiology of Fish. Sheffield: Fish Symp: 1991, 319.

Burzawa-Gérard, E., Baloche, S., Leloup-Hatey, J., Le Menn, F., Messaouri, H., Nunez- Rodriguez, J., Peyon, P., Roger, C., 1994. Ovogénèse chez l'anguille (Anguilla anguilla L.): ultrastructure de l'ovaire a différents stades de développement et implication des lipoprotéines au cours de la vitellogenèse. Bull Fr Pêche Piscic 335, 213-233

Callamand, O., Fontaine, M., 1942. L’Activité thyrodienne de l’Anguille au cours de son development. Arch.Zool.Exp.Gen. 82, 129-135.

Chakraborti, P., Bhattacharya, S., 1984. Plasma thyroxin levels in freshwater perch: influence of season, gonadotropins, and gonadal hormones. Gen.Comp.Endocrinol. 53, 179-186.

Dekker, W., Van Os, B., van Willigen, J., 1998. Minimal and maximal size of eel. Bull Fr Pêche Piscic 0, 195-197

Dickhoff, W.W., Folmar, L.C., Gorbman, A., 1978. Changes in plasma thyroxin during smoltification of coho salmon, Oncorhynchus kisutch. General and Comparative Endocrinology 36, 229-232.

Dufour, S., Delerue-Le Belle, N., Fontaine, Y.A., 1983a. Development of a heterologous radioimmunoassay for eel (Anguilla anguilla) gonadotropin. Gen Comp Endocrinol 49, 403-413

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Dufour, S., Le Belle, N.D., Fontaine, Y.A., 1983b. Effects of steroid hormones on pituitary immunoreactive gonadrotropin in European freshwater eel, Anguilla anguilla L. Gen comp Endocrinol 52, 190-197

Durif, C., Dufour, S., Elie, P., 2005. The silvering process of Anguilla anguilla: a new classification from the yellow resident to the silver migrating stage. J Fish Biol 66, 1025-1043

Etienne, N. 1959. Influence de la maturation sexuelle provoquée sur l'activité thyroïdienne de l'Anguille européenne mâle, Anguilla anguilla L. Société de Biologie, 41-44.

Evans D.H. (ed), 1993. The Physiology of fishes, Vol. CRC Press, Boca Raton, Florida

Fontaine, M., 1975. Physiological mechanisms in the migration of marine and amphihaline fish. Advances in Marine Biology 13, 241-355

Han, Y.S., Liao, I.C., Tzeng, W.N., Huang, Y.S., Yu, J.Y.L, 2003. Serum estradiol-17β and testosterone levels during silvering in wild Japanese eel Anguilla japonica. Comp.Biochem.Physiol. B 136, 913-920.

Han, Y.S, Liao, I.C., Tzeng, W.N., Ju, J.Y., 2004. Cloning of the cDNA for thyroid stimulating hormone β subunit and changes in activity of the pituitary-thyroid axis during silvering of the Japanese eel, Anguilla japonica. Journal of Molecular Endocrinology 32, 179-194.

Huang, Y.S, Rousseau, K, Le Belle N, Vidal, B., Burzawa-Gérard, E., Marchelidon, J., Dufour, S., 1998. Insulin-like growth factor-I stimulates gonadotropin production from eel pituitary cells: a possible metabolic signal for induction of puberty. Journal of Endocrinology 159, 43-52

Jackson, L.F., Sullivan, C.V., 1995. Reproduction of White Perch: the Annual

Gametogenic Cycle. Transactions of the American Fisheries Society 124, 563-577.

Knowles, F., Vollrath, L., 1966. Changes in the pituitary of the migrating European eel during its journey from rivers to the sea. Zeitschrift für Zellforschung 75, 317-327.

Lam, T.J., 1959. Environmental influences on gonadal activity in fish. In: Fish Physiology vol IXB, Behaviour and Fertility Control, Chapter 2, pp.65-116. Edited by W.S.Hoar, D.J.Randall & E.M.Donaldson

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Lokman, P.M., Vermeulen, G.J., Lambert, J.G.D., Young, G., 1998. Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels (Anguilla dieffenbachi and A.australis) before and at the onset of the natural spawning migration. I. Females. Fish Physiology and Biochemistry 19, 325-338.

Lokman, P.M., Detlef, H.R., Davie, P.S., Young, G., 2003. The physiology of silvering in Anguillid eels: androgens and control of metamorphosis from the yellow to silver stage. In: Aida K, Tsukamoto K, Yamauchi K (eds) Eel Biology. Springer Verlag, Tokyo, p 331-349

MacCrimmon, H.R, Claytor, R.R, 1985. Meristic and morphometric identity of Baltic stocks of Atlantic Salmon (Salmo salar). Can.J.Zool. 63, 2032-2037.

Marchelidon, J., Schmitz, M., Houdebine, L.M., Vidal, B., Le Belle, N., Dufour, S., 1996.

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application to the study of silvering and experimental fasting. Gen.Comp.Endocrinol.

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Marchelidon, J., Le Belle, N., Hardy, A., Vidal, B., Sbaihi, M., Burzawa-Gérard, E., Schmitz, M., Dufour, S. 1999. Etude des variations de paramètres anatomiques et endocriniens chez l'anguille européenne (Anguilla anguilla) femelle, sédentaire et d'avalaison: application à la caractérisation du stade argenté. Bulletin Français de la Pêche et de la Pisciculture 355, 349-368

Messaouri-Deboun, H., Baloche, S., Leloup-Hatey, J., Burzawa-Gérard, E. 1991. Hepatic estradiol receptors in European eel (Anguilla anguilla L.) after treatment by estradiol alone or associated with bovine growth hormone. Gen Comp Endocrinol 82, 238

Montero, M., Le Belle, N., King, J.A., Millar, R.P., Dufour, S. 1995. Differential regulation of the two forms of gonadotropin-releasing hormone (mGnRH and cgnRH-II) by sex steroids in the European female silver eel (Anguilla anguilla). Neuroendocrinology 61, 525-535.

Nagahama, Y. 1994. Endocrine regulation of gametogenesis in fish. Int.J.Dev.Biol. 38, 217-229.

Oliverau, M., Ball, J.N. 1970. Pituitary influences on osmoregulation in teleosts. Mem.Soc.Endocrinol. 18, 57-85.

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Romeis, B 1968. Mikroskopische Technik. R.Oldenbourg Verlag, München, Wien, 757 pages.

Rohr, D.H., Lokman, P.M., Davie, P.S., Young, G., 2001. 11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis. Comparative Biochemistry and Physiology, A, 130, 701-714.

Sakamoto, T., McCormick, S.D., Hirano, T., 1993. Osmoregulatory actions of growth hormone and its mode of action in salmonids. A review. Fish Physiology and Biochemistry 11, 15-164.

Sternberg, H., Moav, B., 1999. Regulation of the growth hormone gene by fish thyroid

retinoid receptors. Fish.Physiol.Biochem. 20, 331-339.

Swift, D.R., 1960. Cyclical activity of the thyroid gland of fish in relation to environmental changes. S.Zoological Society London. 2, 17-27.

Tesch, F.W. , 2003. The Eel. Fifth edition, Blackwell Publishing, Oxford

Thioulouse, J., Chessel, D., Dolédec, S., Olivier, J.M., 1997. ADE-4: a multivariate analysis and graphical display software. Statistics and Computing 7, 75-83.

Ueda, H., Hiroi, O., Hara, A.,Yamauchi,. K., Nagahama, Y., 1984. Changes in serum concentrations of steroid hormones, thyroxine, and vitellogenin during spawning migration of the chum Salmon, Oncorhynchus keta. General and Comparative Endocrinology 53, 203-211.

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DIRECT CALORIMETRY OF FREE MOVING EELS WITH MANIPULATED THYROID STATUS.

Vincent van Ginneken1*, Bart Ballieux2, Erik Antonissen1, Rob van der Linden1, Ab Gluvers1, Guido van den Thillart1.

1) Institute Biology Leiden (IBL), Integrative Zoology, Leiden University, The Netherlands. 2) Leiden University Medical Centre, CKCL-laboratory, Leiden University, The Netherlands.

*: Corresponding Author: Dr.V.van Ginneken, Institute of Biology Leiden (IBL), Van der Klaauw Laboratory. POB 9516, 2300 RA Leiden, The Netherlands, FAX: +31(0)71-5274900, TEL: +31(0)71-5277492 E-mail: [email protected]

Submitted to: Naturwissenschaften

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De onreinheid van de paling voor de Joden zoals omschreven in de Bijbel door Antoni van Leeuwenhoek bestreden). Dese twee soorten van vissen sijn de jooden soo sij menen, inde wet Mosis verboden om dat Deuteronomium 14. Vs 10. staat. ‘Maar al wat geen Vinnen en Schobbens heeft en sult gij niet eeten het sal u lieden onreijn sijn. En Leviticus 11. Vs 12. (vande selve vissen sprekende werd geseijt) dat sal u een verfoeijsel sijn.) (Antoni van Leeuwenhoek, Brief No. 81 [42], 25 juli 1684).

Chapter 5

DIRECT CALORIMETRY OF FREE MOVING EELS WITH MANIPULATED THYROID STATUS

Abstract: In birds and mammals, the thyroid gland secretes the iodothyronine hormones of which T4 (Tetrajodothyronine) is less active than T3 (Triiodothyronine). The action of T3 and T4 is calorigenic, and is involved in the control of metabolic rate. Across all vertebrates, thyroid hormones also play a major role in differentiation, development and growth. Although the fish thyroidal system has been researched extensively, its role in thermogenesis is unclear. In this study we measured overall heat production to an accuracy of 0.1 mW by direct calorimetry in free moving European eel (Anguilla anguilla L.) with different thyroid status. Hyperthyroidism was induced by injection of T3 and T4 and hypothyroidism was induced with phenylthiourea. The results show for the first time at the organismal level, using direct calorimetry, that neither overall heat production nor overall oxygen consumption in eels is affected by hyperthyroidism. Therefore, we conclude that the thermogenic metabolism- stimulating effect of thyroid hormones (TH) is not associated with a cold-blooded fish species like the European eel. This supports the concept that TH does not stimulate thermogenesis in poikilothermic species.

Introduction

In endothermic vertebrates (warm-blooded) such as birds and mammals, the thyroid hormones Tetrajodothyronine (T4) and Triiodothyronine (T3) raise the metabolic rate, especially in organs like muscle and to a minor extent in liver, by activating the Na/K ATPase on the cell membrane. In these two groups, the homoeothermic response of the thyroid is regulated via a temperature-sensitive region in the hypothalamus, which further acts on the pituitary followed, by an activation of the thyroid. This system is called the hypothalamic-pituitary-thyroid axis (HPT)-axis. An HPT-axis has been described in several fish species (Leatherland 1988). The main role of thyroid hormones in fish is regulation of growth, development and reproduction (Cyr and Eales 1996). Further, a role in metamorphosis has been suggested for salmon during the parr-smolt transformation (Dickhoff et al. 1978).

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The universal presence of the thyroid gland and of the thyroid hormones T3 and T4, in vertebrates suggests a continuing and common role in ecto- and endotherms. Previous studies have shown that thyroid hormones do not stimulate metabolic rate in poikilothermic species as they do in homeotherms. Rossier et al. (1979) failed to demonstrate an increase in oxygen consumption, Na+ transport, and Na+-K+-ATPase activity following T4 injections in amphibian. For Teleostei, limited data are available at present for the role of thyroid hormones in thermoregulation and regulation and control of metabolic rate (Etkin 1978, Plisetskaya et al. 1983). One study looked at plasma and hepatic nuclear T3 in two lower vertebrates: the lake trout (Salvelinus namacycush) and the sea lamprey (Petromyzon marinus, Weirich et al. 1987). They found that T3 failed to stimulate hepatic oxygen consumption, mitochondrial glycerophosphate dehydrogenase or malic enymze activity in liver trout whereas it did so in rats. While in homeothermic species, thyroid hormones increase thermogenesis, the data in poikilothermic species suggested that it did not. Direct calorimetry in whole, unrestrained poikilothermic animals, such as the eel, would add support to, or challenge this concept. The aim of this study was therefore to assess, with direct calorimetry, the extent to which the thyroid is involved in the control of metabolic rate in a cold-blooded fish species, the European eel.

Material and Methods

Animals and Hormone treatment One year old male eels (Anguilla anguilla L.), total body weight 80-120 grammes, were obtained from a commercial hatchery (Royaal BV., Helmond, The Netherlands). The animals were acclimatised to 20oC and kept under normal laboratory conditions (14 h light, 10 h dar- kness) and normoxic oxygen saturation values of 80%. The animals were fed daily with Trouvit pelleted food (Trouw, Putten, The Netherlands). There were four groups: Control, goitrogenic, T4-treated and T3-treated animals. The goitrogenic group was created by adding Phenylthiourea to the water (0.20 %) for a period of 6 weeks. Phenylthiourea and other thiocarbimides are known goitrogens (antithyroid substances) inhibiting the iodination of tyrosine residues essential for the production of thyroid hormones (Mountcastle 1980). One group (n=10) was treated with T3 while a second group (n=9) was treated with T4.

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In these two groups T3 or T4 (500 μg per ml per 100 g body weight) were dissolved in 0.1 mM NaOH, dissolved in coconut oil (Pradet-Balade et al. 1997) and injected weekly (IP) for a period of one month. Control and goitrogenic eels were also injected weekly with the vehicle (coconut oil) used for T3 and T4 injections.

Calorimeter and Oxygen registration system The calorimetric system and oxygen registration system have been described elsewhere (Addink et al., 1991, van Ginneken et al., 1994). Briefly, it is a differential flow through calorimeter (Sétaram GF 108, Lyon, France), which measures continuously the rate of heat production of the fish in a vessel with a volume of 1 liter. Calibration was performed with a known electrical current and voltage (Sétaram EJ2 joule calibrator) by a current of 3.15 mA with a voltage of 31.64 V which is applied to a resistor of 1000 Ohm mounted in the measurement vessel. This resulted in a power output signal of 99.7 mW. The calibration of every sample point (one per minute) was performed via a 'sensitivity coefficient' using special developed software (van Ginneken et al. 1994). Oxygen consumption was calculated from the difference in oxygen tension between reference and measurement vessel multiplied by the flow through the system (van Ginneken et al., 1994). We determined the Standard Metabolic Rate (SMR) after 24 h as the minimal metabolic rate over a time interval of 1 h (van Ginneken et al. 1997).

Thyroid measurements After taking each eel out of the calorimeter at 70 h after the start of the calorimetry experiment, the fish was rapidly anaesthetized with 200 ppm MS222 (3-aminobenzoic-acid- ethyl-ester methanesulfonate salt, Sigma, St.Louis, USA). Blood was collected from the caudal vein using a heparinized syringe and stored at -80 oC pending further analysis. Total Thyroxin (T4), and Total tri-iodothyronine (T3) were determined by Fluorescence Polarization Immunoassay (FPIA) on an Axsym analyser of Abbott Diagnostics (Hoofddorp, Netherlands)

Statistics Normality of the data and homogeneity of variances were checked by Kolmogorov-Smirnov and

Fmax tests, respectively. When data were normally distributed a one-way ANOVA was used with a Bonferroni-correction. P < 0.05 was considered as statistically significant.

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Results

In Figure 1, a registration of four typical calorimetric experiments (Control, goitrogenic, T4- treated and T3-treated) during a period of 3.5 d. with one eel in the calorimetric vessel are depicted. The experiment starts with a calibration procedure for the heat signal. This results in a heat production of approximately 100 mW. Thereafter, the fish is introduced into the vessel. The oxygen consumption and heat production are measured for approximately 48 h.

The oxygen tension of the in-flowing water is indicated by the top of the spikes in the pO2 signal, which switches every 55 minutes to the reference position. The lowest heat production and concomitantly the lowest oxygen consumption, the Standard Metabolic Rate (SMR), was determined over an interval of 1 h for each eel within the four groups (see Figure 2). For all individual eel of the four groups these are given in Figure 2. Interestingly, despite the goitrogenic animals having low activity levels (which can be concluded from the fluctuations in heat and oxygen signal, see Figure 1B) they had a significantly higher SMR (Table 1, heat production: ANOVA; n=6; P<0.005; oxygen consumption: ANOVA; n=6; P<0.002). In Table 1, the Mean ± SD for T3 and T4 measured in blood plasma, overall heat production (SMR), overall oxygen consumption (SMR) and oxycaloric coefficient for a Control-, Goitrogenic-, T4 and T3 treated European eel (Anguilla anguilla L.) group are given. In the Control group, T4 levels were 4.4 times higher in comparison to T3 levels. Heat production was around 86 J.h-1.100-1 g. oxygen consumption around 0.2 mmol.h-1.100-1 g. while the oxycaloric value was in the range of 433 kJ.mol-1 which corresponds to a mixed substrate (van Ginneken et al. 1994). Our treatment of the eels with phenylthiourea for a period of 6 weeks resulted in a goitrogenic group with significantly lower T3 levels (ANOVA; n=6; P<0.008) in comparison to the Control group. The T4 levels were even zero in comparison to the Control group elevating T3 and T4 levels by dissolving these compounds in coconut oil followed by weekly IP injection during a period of one month was successful. T3 levels increased 6.9-fold over control, while the T4 levels increased 1.4-fold over control. T4 levels increased 53-fold in comparison to the T4 levels in the control group. In the T4 treated group the T3 levels increased 15.9-fold in comparison to the T3 level found in the control group. The most important observation from this study was that heat production and oxygen consumption were not significantly elevated in the T4 (heat production: ANOVA; n=9; P<0.504; oxygen consumption: ANOVA; n=9; P<0.466) and T3 (heat production: ANOVA;

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n=10; P<0.701; oxygen consumption: ANOVA; n=10; P<0.539) treated groups in comparison with the Control group. Only in the goitrogenic group was the metabolic rate significantly increased (heat production: ANOVA; n=6; P<0.005; oxygen consumption: ANOVA; n=6; P<0.002) The oxycaloric value was in the range of 426-433 kJ.mol-1 for all four groups, which corresponds to mixed substrate (van Ginneken et al. 1994).

Figure 1: Registration of 4 typical experiments during a period of 3.5 days to determine the Standard Metabolic Rate (SMR). Each experiment is performed with one eel. A) Control animal (102.9 g), B) Goitrogenic animal (105.9 g), C) T4-treated animal (96.3 g), D) T3- treated animal (93.4 g). The top signal, alternating between reference and measurement vessel of the calorimetric system, is the oxygen tension signal (right Y-axis). The difference in oxygen tension multiplied with the flow through the system gives the oxygen consumption. The lower line is the heat production signal of the fish (left Y-axis). The experiment starts the first 20 h with an electrical calibration until 100 mW and finishes at 72 h after removing the fish from the vessel with an electrical calibration.

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Control Goitro- T4 T3 Co-Goi Co-T4 Goi-T4 Co-T3 Goi-T3 genic (N=6) (N=6) (N=9) (N=10)

T3 2.62 0.61 16.51 17.99 P≤ P ≤ P ≤ P ≤ P ≤ (nmol.l-1) (1.37) (0.54) (6.67) (5.84) 0.008** 0.001** 0.001** 0.001** 0.001** T4 11.64 0 617.85 41.56 P ≤ 0.166 P≤ P≤ P≤ 0.033* P ≤ (nmol.l-1) (19.08) (0) (7.72) (27.06) 0.001** 0.001** 0.002** Heat 86.32 110.52 89.75 88.64 P ≤ P ≤ 0.504 P ≤ P ≤ 0.701 P ≤ (J.h-1.100 g-1) (11.68) (11.55) (7.81) (11.35) 0.005** 0.001** 0.002**

Oxygen 0.20 0.27 0.21 0.21 P ≤ P ≤ 0.466 P ≤ P ≤ 0.539 P ≤ (mmol.h-1.100-1 (0.03) (0.02) (0.02) (0.03) 0.002** 0.001** 0.001** g) Oxycaloric 433.36 430.06 431.35 426.22 P ≤ 0.671 P ≤ 0.801 P ≤ 0.881 P ≤ 0.353 P ≤ 0.641 value (11.02) (14.86) (16.79) (15.95)

Table 1: Mean ± SD for 3,5,3’-triiodo-L-thyronine (T3) and L-thyroxine (T4), heat production, oxygen consumption and oxycaloric coefficient for a Control-, Goitrogenic-, T4 and T3 treated European eel (Anguilla anguilla L.) group. *) denotes significant difference (p≤0.01); **) denotes significant difference (p≤0.001).

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Figure 2: Heat production (Mean ± SD) of a Control, goitrogenic, T3 (2A) and T4 (2B) treated individual European eels (Anguilla anguilla L.) measured by direct calorimetry. Heat production was measured via direct calorimetry with a Setaram, 1 liter, flow-through twin detection microcalorimeter (van Ginneken et al. 1994).

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Discussion

The procedure to elevate T3 and T4 levels by using a vehicle of coconut oil was successful. T3 levels increased 6.9-fold versus controls while even the T4 levels increased 1.4- fold after T3 treatment in comparison to control T4 levels. The interaction between the thyroid hormones (T3 & T4) thyroid stimulating hormone (TSH) and the direct effect of this on the pituitary level, and its indirect effect via modulation of hypothalamic mediators such as thyrotropin-releasing hormone remains a complicated matter (Pradet-Balade et al. 1997). T4 levels increased 53-fold in the T4 treated group compared to controls while there was a 15.9-fold increase of T3 plasma levels. The latter observation can possibly be explained by peripheral T4 deiodination. This observation was also made by Pradet-Balade et al. (1997) for European eel treated with T4. The increased T4 levels in T3 treated eels is not the consequence of the cross- reactivity of the assay. The T4 assay has less than 10% cross reactivity with T3 and the other way around the T3 assay has less than 0.05% cross reactivity with T4 (Pers. Comm. Dr. Bart Ballieux). It is possible that the conversion of T4 into T3 is inhibited by deiodinases as a consequence of the high T3 concentrations resulting in T4 accumulation. In humans the plasma half-life of T4 is 7 times higher than the half-life of T3 and is mainly determined by deiodinase activity. The existence of deiodination pathways in fish is demonstrated in the study of Sweeting and Eales (1992). With conventional techniques like oxygen consumption measurements (indirect calorimetry), some researchers concluded that the energy consumption of cold-blooded animals is not influenced by thyroxine (Bern & Nandi 1964, Gorbman 1969). On the other hand some re- ports show a stimulatory effect of thyroid hormones on oxygen consumption in fishes (Ruhland 1969, Pandey and Munshi 1976) while anti-thyroid treatment rapidly decreased oxygen consumption (Ruhland 1969). Differences between the results of different researchers can possibly be ascribed to methodological flaws in the experimental set up including stress of the animals. Therefore, direct calorimetry in whole, unrestrained poikilothermic animals, such as the eel, would add support to, or challenge this concept. Measuring heat production via direct calorimetry under aerobic conditions has two major advantages: 1) the outcome is dependent on the type of metabolism (anaerobic processes or synthesis. 2) the secondary advantage is that direct heat measurements can possibly elucidate if there is a central regulator (heat-center) in the regulation of body temperature, even in a cold- blooded fish like the European eel.

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We thus measured, with a flow-through twin detection calorimeter, the heat-flow and oxygen consumption of eels with low versus high levels of T3 (Figure 2A, Table 1). and T4 (Figure 2B, Table 1). Our results demonstrate no significant difference in heat production or oxygen consumption over a wide range of T4 or T3 levels. Only in the goitrogenic (=low thyroxine) group was the metabolic rate significantly increased (Figure 2A & B, Table 1). This is an observation not directly related to the most important conclusion from this study, namely that heat production and oxygen consumption are not significantly elevated in the T4 and T3 treated groups in comparison with the control group. The increased metabolic rate in the goitrogenic group can possibly be explained by the toxicity of phenylthiourea. In contrast to the data presented in this manuscript, published literature data from eu-, hypo- and hyperthyroidic human subjects (Hortling & Hiisi-Brummer 1959), clearly demonstrate that the Basal Metabolic Rate (BMR) is dependent on the concentration of thyroid hormones. Our results demonstrate that, despite the universal presence of the thyroid gland and of T3 and T4 in all vertebrates, their calorigenic function is not universal. The study of Hortling & Hiisi-Brummer (1959) support the view that the ability of thyroid hormones to stimulate thermogenesis is solely restricted to the homeothermic species (birds and mammals), which originated more than 200 million years ago (Freake and Oppenheimer 1995). Our observation, reported here, that thyroxine is not calorigenic in eels, suggests that they lack a mechanism to produce obligatory heat. These findings likely explain the lower metabolic rate of poikilothermic species.

LITERATURE

Addink, A.D.F.; van den Thillart, G.; Smit, H.; van Waversveld, J. (1991). A novel 1 liter flow-through calorimeter for heat production measurements on aquatic animals without stress. Thermochimica Acta 193: 41-48. Bern, H.; Nandi, J.(1964). Endocrinology of poikilothermic vertebrates. In: The Hormones, edited by G.Pincus, K.Thimann, and E.B. Astwood. New York: Acad.Press, 1964, vol.4, p.199-299. Cyr, D.G.; Eales, J.G. (1996). Interrelationships between thyroidal and reproductive endocrine systems in fish. Reviews in Fish Biology and Fisheries 6: 165-200.

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Dickhoff, W.W.; Folmar, L.C.; Gorbman, A.(1978). Changes in plasma thyroxine during smoltification of Coho Salmon, Oncorhynchus kisutch. General and Comparative Endocrinology 36: 229-232. Etkin. W.(1978). The thyroid-a gland in search of a function. Perspective in Biology and Medicine: 22(1):19-30. Freake, H.D.; Oppenheimer, J.H. (1995). Thermogenesis and thyroid function. Ann.Rev.Nutr. 15: 263-291. Ginneken van, V.J.T., Gluvers, A., van der Linden, R.W., Addink, A.D.F. and van den Thillart, G.E.E.J.M. (1994). Direct calorimetry of aquatic animals:automated and computerized data-acquisition system for simultaneously direct and indirect calorimetry in aquatic animals. Thermochim. Acta, 247:209-224. Ginneken van, V.J.T. Addink, A.D.F.; van den Thillart, G.E.E.J.M., Körner, F.; Noldus, L.; Buma, M. (1997). Metabolic rate and level of activity determined in tilapia (Oreochromis mossambicus Peters) by direct and indirect calorimetry and videomonitoring. Thermochimica Acta 291: 1-13. Gorbman, A.(1964). Thyroid function and its control in fishes, In: Fish Physiology, edited by W.S. Hoar and D.J.Randall. New York: Wiley, 1969, p. 241-274. Hortling, H.; Hiisi-Brumer, L. (1959). Basal Metabolic Rate and Serum Protein-bound Iodine in Thyroid Disturbances with Special Reference to Goitre and 'Hypometabolism'. Acta Medica Scandinavica 165: 403-411. Leatherland, J.F.(1988). Endocrine factors affecting thyroid economy of teleost fish. Amer.Zool. 28:319-328. Mountcastel, V.B. (1980). Medical Physiology: St. Louis: C.V. Mosby. Pandey, B.N.: Munshi, J.S.D.(1976). Role of the thyroid gland in regulation of metabolic rate in an airbreating siluroid fish, Heteropneustes fossolis (Bloch). J.Endocrinol. 69:421-425. Pradet-Balade, B.; Schmitz, M.; Salmon, C.; Dufour, S.; Quérat, B. (1997). Down- regulation of TSH Subunit mRNA levels by Thyroid Hormones in the European Eel. General and Comparative Endocrinology 108: 191-198. Plisetskaya, E.M.; Woo, N.Y.S.; Murat, J.C.(1983). Review: Thyroid hormones in cyclostomes and fish and their role in regulation of intermediary metabolism. Comp.Biochem.Physiol. 74A:179-187. Rossier, B.C.; Rossier, M.; C.H. Lo (1979). Thyroxine and Na+ transport in toad: role in transition from poikilo- to homeothermy. Am.J.Physiol. 236: C117-C124.

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Ruhland, M.L. (1969). Relation entre l'activité de la glande thyroïde et la consommation d'oxygène chez les Téléostéens, Cichlidés. Experientia 25:944-945. Sweeting R.M. and Eales, J.G. (1992). The Acute influence of ingested Thyroid hormones on hepatic deiodination pathways in the rainbow trout, Oncorhynchus mykiss, General and Comp. Endocrinology 85: 376-384. Weirich, R.T.; Schwartz, H.L.; Oppenheimer, J.H. (1987). An analysis of the interrelationship of nuclear and plasma Triiodothyronine in the Sea Lamprey, Lake Trout , and Rat: Evolutionary Considerations. Endocrinology 120: 664-677.

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Endurance swimming of European eel

G. van den Thillart*, V. van Ginneken, F.Körner, R.Heijmans, R.van der Linden and A.Gluvers

Institute of Biology Leiden,Van der Klaauw Laboratory,POB 9516,2300 RA Leiden, The Netherlands.

Keywords: efficiency;endurance;migration;reproduction;respirometry;swim tunnel.

Published in: Journal of Fish Biology (2004) 65,312–318

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Observatie van schubben bij de paling d.m.v. de Microscoop Dese Huijt soo van Paling en Ael, heb ik gaan ondersoeken en hebbe (nadat ik deselve vande slijm hadde ontbloot) gesien, dat die visschen haar huijt, soo wel met schobbens sijn bedekt, als eenige andere Rivier vis, want de schobbens (al hoe wel deselve seer dun en kleijn sijn) lagen soo oordentelijk en digt int verband, en over malkanderen, als sij in eenige Rivier of Zeevis leggen; daar en boven sijn de Alen en Palingen, soo wel met vinnen versien, als andere visschen, want sij hebben aan ijder sijde van het Hooft, een volkomen vin, en daar en boven is doorgaans haar agterlijf of staart wel onder als boven versien met een doorgaande vin. En omdat dit wat nieuws en voornamentlijk inde jooden haar oogen is (door dien sij tot nog toe in die onkunde hebben geweest dat dese smakelijke vis, haar onreijn en verfoeijlijk was) soo heb ik van de paling een schubbe die ik onder vande Buijk hadde genomen, alwaar die de kleijnste sijn, en geordonneert aan een plaatsnijder, dat soo als hij deselve door het vergroot glas sag, soude af teijkenen. (Antoni van Leeuwenhoek, Brief No. 81 [42], 25 juli 1684).

Chapter 6

ENDURANCE SWIMMING OF EUROPEAN EEL (Anguilla anguilla L.)

ABSTRACT A long-term swim trial was performed with five female silver eels Anguilla anguilla of 0.8 – 1.0 -1 kg (c. 80 cm total length, LT) at 0.5 body lengths (BL) s , corresponding to the mean swimming speed during spawning migration. The design of the Blazka-type swim tunnel was significantly improved, and for the first time the flow pattern of a swim tunnel for fish was evaluated with the Laser-Doppler method. The velocity profile over three different cross sections was determined. It was observed that 80% of the water velocity drop-off occurred over a boundary layer of 20- mm. Therefore, swim velocity errors were negligible as the eels always swam outside this layer. The fish were able to swim continuously day and night during a period of 3 months in the swim tunnel through which fresh water at 19°C was passed. The oxygen consumption rates remained -1 -1 stable at 36.9 ±2.9 mg O2 kg h over the 3 months swimming period for all tested eels. The -1 -1 mean cost of transportation was 28.2 mg O2 kg km . From the total energy consumption the calculated decline in fat content was 30%. When extrapolating to 6000-km this would have been 60%, leaving only 40% of the total energy reserves for reproduction after arriving at the spawning site. Therefore low cost of transport combined with a high fat content are crucial for the capacity of the eel to cross the Atlantic Ocean and reproduce.

INTRODUCTION

In the 1960s and 1970s swimming experiments were performed mainly with salmonids in a stream gutter or swim tunnel (Web, 1971; Brett, 1973). Other studies were carried out in swim tunnels with goldfish Carassius auratus (L). (Bainbridge, 1963; Smit et al, 1971), rainbow trout Oncorhynchus mykiss (Walbaum) (Wood et al., 1983), and carp Cyprinus carpio (Van Dijk et al., 1993). Thus far little information is available about the construction of swim-tunnels even in studies which describe the experimental set-up (Blazka et al., 1960; Brett, 1964; Bell & Terhune, 1970; Smith & Newcomb, 1970). The hydrodynamic aspects of water flow in swim tunnels are rather complex and were never evaluated in experiments with fishes. Brett (1964) optimized the flow pattern in his swim tunnels by inserting small buttons in the flow grids.

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This was done by trial and error (J.R. Brett pers. comm.). The water flow in a tunnel easily becomes turbulent. Laminar flow, which can be reached in long flow tubes, has a wide velocity drop-off zone at the wall in contrast to semi-turbulent flow, which has a short drop-off zone. The width of this zone should be smaller than that of the fish. The double cylinder design of Blazka et al. (1960) enables the construction of large flow tubes requiring much less space (Van Dijk et al., 1993). Eels Anguilla anguilla L. migrate great distances to reach their spawning sites. As silver eels they leave the west coast of Europe in the autums and reach the Sargasso Sea in c. 6 months (Tesch, 1977). The distance they have to cover is c. 6000-km, which means for a 80 cm total -1 length (LT) female eel (1.5 kg) an average swimming speed of c. 0.5 body lengths (BL)s . A characteristic for silver eels is that they stop feeding when they start migrating down the rivers (August to October in the Netherlands). At the start of their journey they are still immature, and their gonads must therefore develop during or after their migration. Eels have considerable quantities of fat as energy stores. As they have to swim a long distance, it is important to know how much of this energy store is needed for swimming and how much for maturation. In order to be able to study the energetics of long-term swimming 22 swim tunnels were constructed. The flow in the swim tunnel was validated by a Laser-Doppler system.

MATERIAL AND METHODS

Swim-tunnel The Blazka-type swim-tunnel used is shown in Fig. 1. The perspex tube has a length of 2000 mm and a diameter of 288 mm. The inner diameter of the swim-tunnel is 190 mm. The mean ± S.D. volume is 127.1 ± 0.9 litre (n=5). The surface area of the inner tube is equal to the surface area of the outer ring (after correction of the wall thickness), such that the water velocity is the same in both compartments. The power of the three-phase electromotors is 400 watt and drives a propeller consisting of three blades of 190.5 mm with a pitch of 177.8 mm. The power is controlled by a Siemens Micro Master (Basic 370) digital power and frequency controller. The motor is equipped with an additional tachometer on the axis for independent (on-line) frequency measurement. The maximal flow is c. 1-m s-1. At the front end of the swim- tunnel a PVC flow streamer is placed with a length of 500 mm, while at the propeller end the flow streamer is 120 mm and the spacing of the streamers is 10 mm. The free space for swimming is 1300 mm.

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At the end of the animal compartment just before the flow streamer, a screen is placed covered with silver wire of 1 mm for electrical stimulation of the fish. The electrical AC current is a sinusoid with a peak of 6.5 V, a frequency of 1 s and duration of c. 20-ms. A magnetic valve controlled the water inlet. When the valve was open the waterflow through each tunnel was set at 5-7 L min-1. After introducing the fish, the waterflow in the inner compartment was corrected for the diameter of the fish according the formulae described by Smit et al (1971). The cross sectional area (A) of the fish was calculated according to: (1) A= π . ½ h . ½w (h= max height, w = max width). The corrected speed is:

(2) Vc = Vm .(1+ Afish/Acylinder), where Vm = measured speed and Vc = corrected speed. From the maximal dimensions of the eels (h=5 cm, w=4 cm) the maximal cross-section is 16- cm2, resulting in a speed correction of c. +5.5%.

Figure 1

Schematic drawing of a 2.0-m swim-tunnel. The tunnel consists of two concentric Perspex tubes of 2 meter and two PVC endcaps. A: electromotor, B: propeller, C: Perspex outer swim-tunnel tube, D: Perspex inner swim-tunnel tube, E: PVC end-streamer, F: animal compartment, G: PVC front streamer. The propeller pushes water into the outer ring and ‘sucks it’ out from the inner tube. The cross-section area of the inner tube and of the outer ring have the same surface area. This results in equal flow rates at both sides. The turbulent water is pushed through streamers that have internal diameters of c. 10 mm.

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Flow measurements The measurements with the Laser Doppler technique were performed at the Delft Hydraulics Laboratory, Technical University Delft. The principle of the Laser Doppler technique is described elsewhere (Drain, 1980; Durst et al, 1981). A 2-dimensional Laser Doppler anemometer (type LDVS02) with a Mean Value Meter (MVM+UCC). The measuring signal of the signal processor (Counter-Tracker) was connected with the Mean Value meter, which integrates and calculates the mean value of the measured analogue signal over a period of 30 seconds. The relation between the revolutions per minute (rpm) of the motor and the water velocity was determined in the centre of the swim-tunnel at 10-cm downstream of the front streamer. The rpm values of the motor were tested in the range of 285-720 rpm. The relation between the measured flow and the rpm value appeared to be linear in this range: (3) Y = 0.0013x – 0.0748 m/s (R2=0.997), where x is rpm. At three positions in the swim-tunnel, at 110, 610 and 1100 mm downstream of the front streamer, the velocity profile was determined over the cross-section of the inner tube. The flow was measured from the wall of the swim-tunnel tube towards the centre at 5, 10, 20, 40 and 95 mm from the wall. The distance of 95 mm from the wall corresponds to the centre of the inner swim-tunnel tube (Fig. 2). The velocity profile was measured at a water velocity of 0.5 m s-1. At 110 mm from the inlet, the water velocity at 40 mm from the wall was still equal to the velocity at the centre. From there the drop-off was steeper than at the other two positions (610 and 1100 mm). The most ideal profile was clearly at 110 mm from the inlet (at the opposite site of the propeller). At the 610 and 1100 mm position the profile at 40 mm from the wall was 15 and 20% respectively below central velocity. So, over the whole length of the animal compartment the velocity at 40-mm from the wall was still close to the set point. This means that animals with a width of > 40-mm swim outside the boundary layer and were thus swimming at the set speed.

Set-up The 22 swim tunnels were placed in the direction of the Sargasso Sea (WNW) in a climatized room of c. 100-m2 (figure 3). The total water content of c. 7000-L was recirculated continuously over a bio-filter. A high capacity protein defoamer, sandfilter, UV-irradiation, and ozoniser were included to improve the water quality. The ozone level was kept low by a feed back control via a redox electrode. The NH3 and NO value of the water was measured daily. At values > 0.1-ppm

NH3 the water was refreshed from a 3000-L tank.

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0.6

0.5

0.4

0.3

Velocity (m/s) 0.2

0.1

0 0 20406080100 Distance to Wall (mm)

Figure 2: The velocity profile from the wall towards the center of the inner tube of the swim tunnel. The flow was measured by a Laser-Doppler system at three cross-sections from the inflow site: ■ 110-mm, υ 610-mm, and ▲1100-mm. At each cross-section the flow was measured at 5 distances from the wall: 5, 10, 20, 40 and 95-mm. The water velocity at the center of the inner tube was 0.5 m/s.

The water temperature was controlled by a separate cooling system. Important parameters such as temperature, salinity, water level, oxygen level, and motor frequency were monitored constantly, logged on a computer, and connected to a telephone alarm system. The data logger was a HP 34970A multichannel logger and controller, equipped with two 40-channel multiplexers (34907A and 34901A). The oxygen electrodes (type Inpro 6415) and the preamplifier (OPA) were from Mettler Toledo (The Netherlands). The illumination in the climatized room was switched to 670-nm light (bandwidth 20-nm) during experiments. Based on pigment changes during silvering, it is assumed that this far-red light is invisible for eels (Pankhorst & Lythgoe 1983). The temperature was kept at 19˚C ,range ±0.5˚C, and the oxygen level between 90 and 75 % air saturation.

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Oxygen consumption The oxygen level in the tunnel was measured continuously by an oxygen electrode (Mettler Toledo). The oxygen consumption rate was calculated from the oxygen decline after automatic closure of the water-inlet by a magnetic valve. The oxygen levels changed between 85 and 75% air saturation. The valve was normally open allowing a refreshment rate of 5-7 L min-1 and automatically operated between 14.00 and 17.00 hours to measure oxygen consumption. From

the decline of the O2-concentration, the oxygen consumption rate was calculated following the formula: -1 -1 (4) VO2 = 127 . Δ[O2]/Δt . fi (mgO2 h kg ), where: Δ[O2]/Δt is the decrease of the oxygen content per hour, fi is the correction factor per 0.8 fish (i), and fi = (Wi/1000) , and M is the mass of the fish (van den Thillart and Kesbeke, 1980).

Animals Eels were obtained from a commercial eel farm Royaal BV, Helmond, the Netherlands. Before transfer into the swim tunnels seven eels were anaesthetised with 200 ppm MS222 (tricaine methanosulphonate, Sandoz). Immediately after losing equilibrium they were placed in the swim tunnels, where they recovered quickly. The flow in the swim tunnels was set at low speed to let the animals habituate to the new environment. The next day the speed was increased stepwise by 0.1 m s-1 per hour to 0.5 BL s-1. Most eels were willing to follow the water current, two were taken out because of poor swimming performance, the others appeared to be good sustained swimmers. The swim tunnels were positioned in the direction of the Sargasso Sea to have a corresponding earth magnetic field direction. For every individual eel the water velocity was set at 0.5 BL s-1. So, the smallest animal of 69.5-cm swam 30.0-km per day and the largest animal 32.4-km per day.

RESULTS

The five eels in this study did not show any disturbance or fatigue during the swim trial of 3 months. The length, body weight, and oxygen consumption rates of the individual swimmers are presented in Table I. The animals swam day and night from March until July for a period of 95 days. The distance covered by the eels was 95x 30=2850-km. The oxygen consumption rates -1 -1 remained constant throughout the 3 months period between 30 and 50 ml O2 kg h . The oxygen consumption pattern over a period of 3 months is shown in Fig. 3. The individual lines

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in Fig. 3 show the variability in oxygen consumption, which differed sometimes by 15 mg O2 kg-1 h-1 on a day to day basis. The behaviour of the animals was rather quiet. The differences in oxygen consumption rates could not be related to visual differences in swimming pattern. At a -1 -1 -1 mean swimming speed of 1.4-km h and an oxygen consumption rate of 36.9 mg O2 kg h , -1 -1 the mean cost of transportation over 2850-km was 36.9 divided by 1.4 = 26.4 mg O2 kg km .

DISCUSSION

The principle of the Blazka swim-tunnel had been given in earlier publications (Blazka et al., 1960; Smith & Newcomb, 1970; Van Dijk et al, 1993) but flow characteristics of a Blazka-type swim-tunnel as far as is known, has never been described before. In this study the very accurate Laser-Doppler system was used to demonstrate the homogeneity of the flow in the swim tunnels. The actual flow was measured at different cross-sections and at different distances from the wall. A linear relationship was observed between the number of rpm and the measured water velocity. The linearity existed up to 0.9 m s-1. The flow between 40-mm from the wall to the centre stayed within a few percent of the setpoint. So, fish with a width of > 40-mm can not swim in the boundary layer. The eels used in this study needed an even wider space because of the amplitude of their tail beat. Furthermor, the head of swimming eels remained between 50 and 100-mm from the wall.

The long-term swim experiments with 5 eels of c. 0.9-kg indicated that eels can be forced to swim under laboratory conditions for a very long period without resting. Five out of seven eels were able to swim for 3 months at 0.5 BL s-1, covering a distance of 2850-km. The applied swimming speed was deduced from the scarce data on spawning migration of A. anguilla.

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Table 1: Data on 5 individual eels that swam continuously at a speed of 0.5 BL/sec for 95 days in 2.0-m swimtunnels.

Fish Number LT Body Weight Oxygen Consumption -1 -1 (mg O2 kg hour ) (cm) (g) Mean (± SD) 1 69.5 854 34.1 ± 5.5 2 74.0 857 36.6 ± 4.8 3 75.0 953 40.3 ± 3.1 4 74.0 907 34.1 ± 5.2 5 71.0 1025 39.4 ± 6.8 Mean ± SD 72.7 ± 2.1 919 ± 64 36.9 ± 2.9

60

50

40

30

O2-consumption 20

10

0 0 20406080100 days

-1 -1 Figure 3: The oxygen consumption (mg O2.kg .h ) rates of 5 adult eels of c. 1-kg. The eels were swimming continuously at 0.5 BL s-1 and at 19°C during a period of 95 days. Details of individual animals used in this experiment are given in Table I.

If the female eels leave the European coasts in September to October and the smallest lepthocephali larvae are observed in the Sargasso Sea in the period February to June (Schmidt, 1923; Fricke & Kaese, 1995), the migrating silver eels have to cover a distance of 6000-km in < 6 months. This is 33-km per day or a mean swimming speed of 0.39 m s-1, corresponding to 0.5 BL s-1 for 80-cm eels.

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There are limited data available on swimming performance of other anguillids (Webb, 1975; McCleave, 1980). The swimming movement of eel is less efficient than that of for example salmonids (Videler, 1993; Bone et al 1995). Biomechanical efficiency is, however, different from overall efficiency. The latter is expressed in J kg-1 km s-1, which is for the energy budget of the animal the most relevant measure. Based on a 10 day swim trial with A.anguilla silver eel the energy costs of swimming of those eels was found to be extremely low: 0.58 J g-1 km-1 (Van Ginneken & Van den Thillart, 2000). This is 2.4-3.0 times lower than values reported in literature for other fish species (Schmidt-Nielsen, 1972). Eels have a fat content of 10-28% with a mean of 20% (Svedäng & Wickström, 1997), which is obviously the predominant energy store. From the oxygen consumption rate (table I) the total energy consumption was 2367 kJ kg-1 for a 3 months period, and the equivalent fat loss was 60.0 g kg-1. Extrapolating from 2850 to 6000-km, the fat consumption for a complete run would have been 126.5 g kg-1. This amount is 60% of the total fat reserve of most silver eels, assuming 200 g fat kg-1. Thus animals < 13% fat would not be able to reach their spawning site. Obviously a low cost of swimming combined with a fat content > 13% is crucial for the capacity of the eel to swim across the ocean.

ACKNOWLEDGMENTS This study was supported by the Dutch Foundation for Technical Research (NWO-STW # LBI66.4199). The authors are very grateful for the help of P. Niemantsverdriet, P. Snelderwaard, S. v Schie, and E. Anthonissen.

REFERENCES

Bainbridge, R. (1963). Caudal fin and body movements of some fish. Journal of Experimental Biology 40, 23-56. Bell, W.H., & Terhune, L.D.B. (1970). Water tunnel design for fisheries research. Journal of the Fisheries Research Board of Canada, Technical Reports 195:69 p. Blazka, P., Volf, M., & Ceplea, M. (1960). A new type of respirometer for determination of the metabolism of fish in an active state. Physiol Bohemoslov 9, 553-560. Bone, Q., Marshall, N.B., & Blaxter, J.H. (1995). Biology of fishes. 2-nd ed, Chapman & Hall, London. ISBN 0-7514-022.

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Brett, J.R. (1964). The respiratory metabolism and swimming performance of young sokeye salmon. Journal of the Fisheries Research Board of Canada, 21, 1183-1226. Brett, J.R. (1973). Energy expenditure of Sockeye Salmon Oncorhynchus nerka during sustained performance. Journal of the Fisheries Research Board of Canada 30, 1799-1809. Drain, L.E. (1980). The Laser Doppler Technique. Wiley & Sons, New York. ISBN 0-471- 27627-8. Durst, F., Melling, A., & Whitelaw, G.H. (1981). Principles and Practice of Laser-Doppler Anemometry. Academic Press, 2nd ed, London. ISBN-012-225260-8. Fricke, H., & Kaese, R. (1995). Tracking of artificially matured eels (Anguilla anguilla) in the Sargasso Sea and the problem of the Eel's Spawning Site. Naturwissenschaften 82, 32-36. McCleave, J.D. (1980). Swimming performance of European eel (Anguilla anguilla L.) elvers. Journal of Fish Biology 16, 445-452. Pankhurst, N.W., & Lythgoe, J.N. (1983). Changes in vision and olfaction during sexual maturation in the European eel Anguilla anguilla (L.). Journal of Fish Biology 23,229-240. Schmidt, J. (1923). Breeding places and migration of the eel. Nature 111, 51-54. Schmidt-Nielsen, K. (1972). Locomotion: Energy Cost of swimming, flying, and running. Science 177, 222-228. Smit, H., Amelink-Koutstaal, J.M., Vijverberg, J., & Von Vaupel-Klein, J.C. (1971). Oxygen consumption and efficiency of swimming goldfish. Comp Biochem Physiol 39A, 1-28. Smith, L.S., & Newcomb, T.W. (1970). A modified version of the Blazka respirometer and exercise chamber for large fish. Journal of the Fisheries Research Board of Canada 27, 1321- 1324. Svedäng. H. & Wickström, H. (1997). Low fat contents in female silver eels: indications of insufficient energetic stores for migration and gonadal development. Journal of Fish Biology 50, 475-487 Tesch, W.W. (1977). The eel. Biology and management of anguilled eels. Chapman & Hall, London, 434 pp. Van den Thillart, G., & Kesbeke F. (1980). Anaerobic production of carbon dioxide and ammonia by goldfish, Carassius carassius (L.). Comp Biochem Physiol 59A, 393-400. Van Dijk, P.L., Van den Thillart, G., Balm, P., & Wendelaar Bonga, S. (1993). The influence of gradual water acidification on the acid/base status and plasma hormone levels in the carp. Journal of Fish Biology 42, 661-671.

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Van Ginneken, V., & Van den Thillart, G. (2000). Eel fat stores are enough to reach the Sargasso. Nature 403, 156-157. Videler, J.J. (1993). Fish Swimming. Chapman & Hall, London, Fish and Fisheries Series 10, 260 pp. Webb, P.W. (1971). The swimming energetics of trout. II. Oxygen consumption and swimming efficiency. Journal of Experimental Biology 55, 521-540. Webb, P.W. (1975). Hydrodynamics and energetics of fish propulsion. Journal of the Fisheries - Research Board of Canada 190, 77-105. Wood, C.M., Turner, J.D., & Graham, M.S. (1983). Why do fish die after severe exercise? Journal of Fish Biology 22, 189-201.

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Acute stress syndrome of the yellow European eel (Anguilla anguilla Linnaeus) when exposed to a graded swimming-load

Vincent J.T. van Ginneken, Paul Balm, Vinod Sommandas, Marjolijn Onderwater and Guido van den Thillart

Institute of Biology Leiden,Van der Klaauw Laboratory,POB 9516,2300 RA Leiden, The Netherlands.

Published in: Netherlands Journal of Zoology (2002) 52(1): 29-42.

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Over de productie van huidslijm door de paling Vorders heb ik gaan examineren de Materie die dese Schobbens bedekt, welke men slijm noemt, en meest doorgaans geoordeelt wert maar een slijm te wesen, die dese vis van buijtenen aan komt; maar ik heb ter contrarie ondervonden, ende seer naakt gesien, dat dese soo genoemde slijm niet van buijten aangroeijt maar dat het waarlijk een gedeelte van het Lighaam selfs is, door dien dese materie (hoewel dat deselve int oog, en door een Microscope ook veeltijds sodanig te voren komt als een Christalline vogt) niet anders en is, dan dooreenlopende Aderkens, die van een ongeloofelijke dunte sijn, en die haar in soo een onbegrijpelijke groote meenigte van takjens, die door malkanderen verspreijt leggen, haar uijtspreijen, dat ik deselve niet dan met de grootste verwondering heb aanschout, ja veele waren soo dun, dat ik die niet als met de aldernaauwkeurigste opmerkinge konde bekennen; en daarenboven imagineerde ik mijn datter nog kleijnder vaatgens waren, die ik niet distinct konde sien. (Antoni van Leeuwenhoek, Brief No. 88 [47], 12 october 1685).

Chapter 7

ACUTE STRESS SYNDROME OF THE YELLOW EUROPEAN EEL (ANGUILLA ANGUILLA LINNAEUS) WHEN EXPOSED TO A GRADED SWIMMING-LOAD

ABSTRACT

In a Blazka swim tunnel (length 170,0 cm; outer diameter swim-tunnel tube 28,8 cm and an inner diameter of 19,0 cm) short-term swim experiments with groups of 120 g eel (≈ 40 cm) at different swimming velocities varying from 0.25 until 3 body lengths (BL) per second were performed. In these experiments, substrates (FFA, glucose), the stress hormone cortisol, parame- ters from the ionic balance (sodium, potassium and chloride) and lactic acid were measured in the blood plasma at 0 (control group), 0.25, 0.5, 0. 75, 1, 1.5, 2.0, 2.5 and 3.0 BL/sec. It is concluded that a swimming speed up to 2 BL/sec is not stressful for yellow eel because the ionic balance is maintained, there is no evidence for activation of the pituitary-interrenal axis and the anaerobic metabolism is not activated. However swimming performances above 2 BL/sec showed a dichotomous pattern: some animals showed no changes while others showed the 'acute stress syndrome' resulting in elevated cortisol levels, glucose mobilization, ionic imbalance and lactate accumulation. Based on these observations it can be concluded that eel-like (anguilliform) swimming is more suitable for long term sustained swimming than for burst activity

INTRODUCTION

In the 1960s and 1970s some swimming experiments were performed with salmon in a stream gutter (Brett 1965, 1967, 1973; Brett and Glass 1973), with respectively, goldfish (Smit 1965, Smit et al. 1971), trout (Webb 1971) and mackerel (Hunter et al. 1971) in swim-tunnels. Only recently the research with swim-tunnels or similar devices was continued in work on plaice (Priede and Holliday 1980), trout (Johnston and Moon 1980, Wood et al. 1983, Ristori and Laurent 1985), salmon (Virtanen and Forsman 1987), Arctic charr (Christiansen and Jobling 1990) and carp (van Dijk et. al. 1993). The latter study for example (performed at our laborato- ries) demonstrated that the combination of water acidification and labor works synergistically (van Dijk et al. 1993).

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Stress induction by exercise may be an interesting research topic exclusively suitable for migrating anadromic fish species such as salmon, catadromic fish species such as eel and ocean rangers such as tuna (Smith 1985). The resulting physiological and endocrinological changes observed in fish after exposure to exercise can only be studied systematically in laboratory studies. Limited studies on the swimming performance of eels or other anguilliform swimming teleosts are available (Webb 1975, McCleave 1980, Müller et al. 1995). In particular, data at low swimming speeds (<3Bl/sec) for eels are lacking despite the indications that cruising speeds at this level are preferred by this fish species (see discussion). Earlier, McCleave (1980) tried to obtain swimming data of elvers at speeds of 3 BL/sec or below. He did not succeed because of the characteristic 'searching for shelter behavior' of eel. Recently two kinematic studies with eel emerged, studying the pattern of undulatory locomotion in water and on land (Gillis 1998), and the changes in muscle performance between yellow and silver eel (Ellerby et al. 2001). Little is known about swimming performance and maximum swimming speed of yellow and silver eel. Because it recently became clear by the work of Ellerby et al. (2001) that there are changes in muscle performance prior to migration, we hope in future studies to extend this work with similar studies on silver eel. The objective of this study was to examine the swimming performance of yellow eel at different swimming speeds in order to determine the range of the aerobic scope. We hypothesize that at intermediary swimming velocities in the range of the aerobic scope, metabolite and ionic levels will remain constant in the blood, but at a certain threshold there is a collapse of the animal resulting in changes of primary and secondary stress parameters in the blood. This threshold point when anaerobic metabolism becomes activated is not strictly defined among animals and will depend on factors such as respiration capacity, energy stores, and capacity for end product elimination of individuals. Therefore, an individual variation of the aerobic scope among animals may be expected. To test this hypothesis we investigated in this study the sustained swimming speed (which is defined as the swimming speed at which an animal is capable of swimming at that speed for hours (Hunter 1971)) of yellow eel from a hatchery. Short-term swim experiments with groups of 120 g eel (≈ 40 cm) were performed in a Blazka swim tunnel at different swimming velocities varying from 0.25 until 3 body lengths (BL) per second were performed. In order to avoid the problems of McCleave (1980) with eels refusing to swim, the animals were forced to swim by an electrical grid at the bottom end of the tunnel. In the performed experiments, substrates (FFA, glucose), the stress hormone cortisol, parameters

111 Chapter 7 from the ionic balance (sodium, potassium and chloride) and lactate which is an indicator of an activation of the anaerobic metabolism, were measured in the blood plasma at several levels of exercise. In this study with yellow eel, we investigated at which swimming velocities the animals remained energetically in homeostasis and at which swimming velocity the aerobic scope is ended resulting in a collapse of the animals with elevated cortisol levels, glucose mobilization, ionic imbalance and lactate accumulation. Based on these observations we can determine the sustained swimming speed of yellow eel.

MATERIAL AND METHODS

Animals: Eels were obtained from a commercial eel farm Royaal BV, Helmond, the Netherlands. At the moment of selection, fish aged from 1-3 years. In the laboratory the animals were kept for one month with a 14:10 light-dark cycle in running local tap-water in aquaria at 19 ± 1 oC and were fed daily with Provimi pelletted food (Provimi, Rotterdam, The Netherlands). The day before the experiment the animals were weighed, their length was measured and they were placed in a group of six to seven animals in the swim-tunnel, which was supplied, with a fresh water supply of 20 l per min. The mean body length of the total group, corresponding to 39.6 ± 5.6 cm, was determined and was defined as 1 body length (1 BL) while the bodyweights were 118.2 ± 15.38 grams.

Blazka swim-tunnel: The Blazka swim-tunnel was calibrated with a Laser Doppler technique at the Delft Hydraulics Laboratory, Technical University Delft. The Blazka swim-tunnel is given in detail in figure 1.

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Figure 1: Blazka swim tunnels used in the experiments with yellow eel. It has a length of 170,0 cm, an outer swim-tunnel tube diameter of 28,8 cm and an inner swim-tunnel tube diameter of 19,0 cm. The power of the engine is 400 watt while the propeller consists of three blades of 7.5 inch with a pitch of 7 inch. At the top end of the swim- tunnel is placed a PVC flow conditioner with a length of 60 cm while at the propeller-end a flow conditioner is placed of 20 cm. The swimming compartment for the fish is 90,0 cm. At the bottom end of the tunnel a screen is placed of plaited silver wire of 1-mm thickness to conduct the electrical current to stimulate the fish to swim. The electrical current is a sinusoid with a peak of 10 V with a frequency of 1 sec. The Blazka swim- tunnel has earlier been depicted elsewhere (van Dijk et al. 1993b ).

Experimental protocol and sampling procedure: We selected the following swimming speeds for the sustained swimming effort for a period of 6 hours: 0 (control group), 0.25, 0.5, 0.75, 1, 1.5, 2.0, 2.5 and 3.0 BL/sec. Fish were placed the evening before the day of the swimming effort in the swim-tunnel at 19 oC. After an introductory period of about 30 minutes at low speed (0.2 BL/sec) the fish were forced to swim at the selected

113 Chapter 7 swimming speed for a period of 6 h. Because it is known that eels do not swim voluntary in swim tunnels (see McCleave 1980), an electrical grid was placed at the bottom end of the tunnel to stimulate the fish to swim.

After exposure to the exercise protocol, the fish were quickly anaesthetized with 300 PPM MS222 (3-aminobenzoic-acid-ethyl-ester methanesulfonate salt, Sigma, St. Louis, USA). After 3 minutes the anaestethized fish were taken out of the swim-tunnel and blood was collected with a heparinized syringe (flushed with 3000 units heparin per ml blood). Above 2 BL/sec we observed in incidental cases that some eels showed signs of fatigue and refused to swim. Those eels were directly removed from the tunnel via a special entry with valve at the bottom end of the Blazka respirometer. Thereafter, the animals were checked macroscopically for parasites on gills and in the swim bladder, or for signs of injury or external wounds. All animals looked healthy. Therefore, we can conclude that according to our initial expectation (see introduction) the threshold point when anaerobic metabolism becomes activated and the animals ultimately collapse will show an individual variation among animals for the factors ‘swimming speed’ and period of ‘swim exercise’. Those animals, which stopped swimming were called the 'exhausted' group (see table 1). Since in the 'exhausted group' (except for lactate, see discussion) no trends and/or significant differences were observed between the different swimming velocities or swimming endurance periods (see table 2), data were pooled. Because at 3.5 BL/sec all animals collapsed and stopped swimming only data for ‘exhausted’ group were available at this swimming speed (table 1 & 2).

Analytical methods blood sample: In the freshly collected blood samples, treated with anticoagulant, haemotocrit was measured directly in 9 μl whole bloodsample using a haematocrit micro-centrifuge (Bayer, F.R.G.). Haemoglobin content in 20 μl blood was detected after 3 minutes using the cyanmet- haemoglobin method (Boehringer Mannheim, F.R.G.). Blood was directly centrifuged (10,000 rpm for 5 min). The plasma was divided in eppendorf tubes (50, 40, 50, 50, 33, 33 μl for respectively cortisol, FFA, glucose, lactate and sodium, potassium and chloride analysis) and stored at -80oC pending analysis. For the glucose measurements, 50 μl plasma was mixed with 200 μl 6% trichloric acid solution to precipitate plasma proteins and stored at -80oC. Glucose was determined by colorimetric assay (Sigma, St.Louis, U.S.A.). Cortisol was measured by radioimmunoassay (Balm et al. 1994). FFA was measured with a commercial test-kit WAKO

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(NEFA C method, Instruchemie, Hilversum, The Netherlands). Lactic acid was determined with an enzymatic test-combination of Boehringer Mannheim: 139084 for L-lactate. Plasma sodium, potassium and chloride levels were measured by flame photometric and colorimetric procedures (Technicon) (Balm & Pottinger 1993). Calculations and statistics: The mean value of every group (swimming velocity) was compared to the mean value of the control group (table 1). Mean ± SD are given in the tables. Statistics were performed using a one-way ANOVA. Comparisons of mean squares of the ANOVA were tested using F-tests. P≤ 0.05 was considered as statistically significant. Normality of the data and homogeneity of

variances were checked by Kolmogorov-Smirnov and Fmax tests, respectively.

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Co (n=6) 0.25 (n=6) 0.5 (n=6) 0.75 (n=6) 1.0 (n=7) 1.5 (n=7) 2.0 (n=6) 2.5 (n=6) 3.0 (n=6) Exh (n=8) Haematocrit 37.68 39.65 40.65 37.02 38.58 36.25 40.53 37.98 36.33 35.78 (%) (4.80) (4.26) (4.17) (5.62) (4.28) (5.30) (5.52) (2.17) (2.30) (3.86) Haemoglobin( 7.02 7.89 7.24 6.37 6.86 6.17 6.90 7.00 6.87 4.92 mM) (1.30) (1.10) (0.67) (0.94) (0.56) (0.57) (0.42) (0.55) (0.26) (0.92) Plasma glucose 3.61 4.49 4.23 4.34 4.30 3.00 4.28 4.51 5.57 11.23* (mM) (1.10) (1.44) (1.16) (1.24) (1.68) (1.52) (1.46) (0.93) (1.57) (2.30) Plasma lactate 0.48 0.35 0.39 0.24 0.52 0.17 * 0.16 * 0.36 0.21 4.13* (mM) (0.08) (0.07) (0.07) (0.05) (0.11) (0.03) (0.04) (0.06) (0.06) (1.73) Plasma cortisol 58 61 81 49 35 40 81 34 35 25 (ng/ml) (23) (14) (20) (34) (9) (16) (26) (24) (26) (19) Plasma FFA 0.214 0.239 0.363 0.522 0.593 0.507 0.283 0.404 0.475 0.236 (mM) (0.107) (0.016) (0.068) (0.189) (0.159) (0.159) (0.045) (0.085) (0.127) (0.107) Plasma sodium 143.5 176.8 131.1 141.1 146.4 144.5 158.9 173.7 142.5 149.0 (meq/l) (15.0) (17.2) (10.8) (18.4) (33.8) (12.0) (19.6) (24.8) (7.2) (13.2) Plasma 2.92 3.54 2.60 2.44 2.66 2.10 2.88 2.76 2.12 4.66* potassium (0.60) (0.60) (0.40) (0.40) (0.60) (0.20) (0.40) (0.60) (0.40) (0.71) (meq/l) Plasma 92.3 116.8 89.0 91.1 95.5 91.8 100.3 108.5 92.1 102.8 chloride (17.6) (15.2) (9.4) (12.0) (21.4) (11.6) (15.0) (15.4) (8.0) (20.6) (meq/l)

Table 1: Parameters measured in plasma of yellow eel (Anguilla amguilla L.) in a Control group (Co), exhausted group (Exh) and at different swimming velocities (0.25, 0.5, 1, 1.5, 2, 2.5, 3 Body Lengths per second, with one bodylength is 39.6 ± 5.6 cm). The mean value of six to eight animals is given with the standard deviation between brackets. * denotes significant difference (P ≤ 0.05) from control group.

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Animal Time Swimming Haemato Haemo- Lactate Glucose FFA Cortisol Na+ K+ Cl- (min) Speed -crit globin (mM) (mM) (mM) (ng/ml) (meq/l) (meq/l) (meq/l) (BL/sec) % (mM) 1 10 2.5 35 3.99 5.59 10.81 0.253 21.8 177.9 5.48 121.2 2 15 2.5 42.2 6.21 5.99 13.54 0.133 15.5 143.6 5.04 102.5 3 30 2.5 36.1 5.67 6.24 14.20 0.266 52.0 148.1 4.92 84.1 4 5 3 32.7 4.74 2.48 9.07 0.375 9.3 135.4 3.82 77.9 5 3 3 41.8 5.45 1.71 9.35 0.129 6.5 151.9 3.46 97.5 6 70 3.5 31.4 5.49 3.35 6.62 0.152 12.3 139.6 4.40 122.8 7 137 3.5 34.5 6.37 4.75 14.51 0.426 31.0 142.0 5.32 84.1 8 165 3.5 32.5 5.41 2.92 8.73 0.156 54.3 153.7 4.80 132.5 Mean - - 35.78 4.92 4.13 11.23 0.236 25.3 149.0 4.66 102.8 STD - - 3.86 1.92 1.73 2.30 0.107 18.8 13.2 0.71 20.6

Table 2: Overview of the composition of the exhausted group

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RESULTS

For the groups between 0 and 3.0 BL/sec, with the exception of plasma lactate at 1.5 and 2.0 BL/sec, no significant differences were observed with the control group for all parameters (table 1). In the 1.5 and 2.0 BL/sec groups plasma-lactate was significantly lower compared to the control group and groups up to 1.0 BL/sec. The metabolism probably is still aerobic and in balance and the decline of plasma lactate can be explained because lactate acts like substrate that can be used by the exercised muscle in situ. Milligan and Wood (1986) demonstrated that more than 80% of the lactic acid generated by the exercised muscle is retained in the muscle mass . So up to a swimming speed of 2 BL/sec the eels are energetically and metabolically in equilibrium and they can perform at this swimming speed without any collapse. Above 2 BL/sec the anaerobic metabolism becomes activated resulting in a rise of plasma lactate. In those groups animals showed (in incidental cases) signs of fatigue and refused to swim from which the ‘exhausted’ group was created. It is clear that in those animals there is a collapse, the aerobic threshold has been passed, the anaerobic metabolism activated, and primary- (cortisol) and secondary- (glucose, lactate, potassium) stress parameters activated. Because there was a large individual variation in the moment of exhaustion, the ‘exhausted group’ consisted of a pooled group of 8 animals as given in table 2.Comparison of the 'exhausted group' with the control group revealed no significant differences for hematocrit, hemoglobin, FFA, cortisol, Na+ and Cl-. Balm et al. (1994) however observed a rapid sampling-associated elevation of cortisol (Balm et al. 1994). This was also observed in this study, giving the following cortisol values ranking according to fish number: 1) 6.61 ± 2.78 (n=9); 2) 47.8 ± 6.9 (n=9); 3) 50.4 ± 12.1 (n=9); 4) 70.5 ± 11.8 (n=9); 5) 73.8 ± 22.1 (n=9); 6) 60.41 ± 22.6 (n=9) (ng/ml). Therefore, it is more accurate to compare the first animal of the groups for plasma cortisol until 2 Bl/sec with the ‘exhausted group’. This gives more information about a stimu- lation of the pituitary interrenal axis. First the cortisol data of 'first sampled' animals were checked with Kolmogorov-Smirnov test for normality. They were normally distributed (P ≤ 0.016). Using this approach a strongly significant difference is observed between the exhausted group (cortisol: 25.34 ± 18.80 ng/ml) and the pooled group of ‘first sampled animals’ (cortisol: 6.61 ± 2.78 ng/ml) (P≤0.003). Gilham & Baker (1985) gave for unstressed and stressed European eel a plasma cortisol concentration of 21.4 vs. 127.6 nmol/l respectively, which corresponds to 7.32 vs. 43.6 ng/ml. Comparing our cortisol data with those values it is clear that the cortisol plasma value of the

118 Chapter 7 pooled group of ‘first sampled animals’ in our study corresponds with the value of Gilham & Baker (1985) for unstressed eels. However the cortisol value found in our exhausted group is half of the value found by those authors in stressed eel (Gilham & Baker 1985). Despite this lower value in our exhausted stressed group, the difference between control (pooled group of first sampled animals) vs. exhausted eel group in our study is significantly different. In addition the rise of cortisol in the exhausted group corresponds with 383 %. Based on these plasma cortisol values we conclude that the eels in the exhausted group were stressed by the experimental protocol (exhaustive exercise). Furthermore significant differences were observed between the control group and exhausted group for lactate (increase 860 %), glucose (increase 311 %) and K+ (increase 160%) (table 2). Those parameters are primarily used as secondary indicators of stress (Wendelaar Bonga, 1997). These results are indicative that up to 2 BL/sec swimming eels are energetically and metabolically in homeostasis. In general, the anaerobic threshold is above 2 BL/sec. Within the selected time interval of 6 hours this leads in incidental cases to a collapse of animals with primary and secondary stress effects. It may be clear that there is an individual variation in the moment of this collapse. We found for individual animals a collapse at 2.5 BL/sec after 10, 15 and 30 minutes respectively, at 3 BL/sec after 3 and 5 minutes, and at 3.5 BL/sec after 70, 137 and 165 min respectively.

DISCUSSION

The most important conclusion from this study is that at intermediary swimming velocities, within the aerobic scope, the swimming eels remain in homeostasis. Similar results were found with cannulated carp (van Dijk et al. 1993a). However, beyond the aerobic scope, at a certain threshold, there is a collapse of the animal resulting in changes of metabolite levels and ion in the blood. This threshold point when anaerobic metabolism becomes activated is not strictly defined among animals and will depend on the aerobic/anaerobic capacity of the animal. Therefore an individual variation of the aerobic scope among animals may be expected in swimming speed and time of exposure before a collapse occurs. The ‘exhausted group’ is created from this pool of collapsed animals. In this respect, it can be questioned if in this way no selection is made for the weakest, injured or parasitized animals. This is partly true. Because we checked the animals macroscopically, injuries or parasites can be excluded, but in this was a selection can be made for the animals with the narrowest aerobic scope and the most limited anaerobic capacity. However,

119 Chapter 7 collapse of these animals occurred all above 2 BL/sec, which doesn’t undermine our main conclusion that the optimal swimming speed for yellow eel from a hatchery is up to 2 BL/sec. From the performed short-term swim experiments with eels of ≈120 g, it is concluded that a swimming speed until 2 BL per second is not stressful for yellow eel because the ionic balance is maintained, there is no evidence for activation of the pituitary-interrenal axis and the anaerobic metabolism is not activated. Above 2 BL per second the animals showed signs of exhaustion which can be associated with the 'acute stress syndrome' resulting in lactate accumulation, elevated cortisol levels, glucose mobilization and ionic imbalance. From studies with other fish species it was concluded that the 50% fatigue-time during a sustained swimming effort was 4 BL per second (Brett 1967) or 3 to 4 BL per second (Blaxter 1969). The energy cost and efficiency for eel-like swimmers may be different compared to other fish species and probably this type of propulsion is more suitable for long distance swimming (see further). The tremendous rise of lactate in plasma in the exhausted group demonstrates that the metabolism became anaerobic and that swimming was strenuous. In the exhausted group it is characteristic that animals which at an earlier moment failed to swim and showed the 'acute stress syndrome' had higher levels of plasma-lactate. Probably the plasma-[lactate] is negatively correlated with the period of swimming. In animals which showed a longer endurance period of activity, plasma-[lactate] was lower. This observation probably can be explained because lactate acts like substrate. More than 80% of the lactic acid generated by the exercised muscle is retained in the muscle mass (Milligan and Wood 1986). Another characteristic of the ‘exhausted group’ is a hyperglycemia. This probably can be attributed to glycogenolytic effects of catecholamines on the liver (Nakano and Tomlanson 1967, Mazeaud et al. 1977). Those hormones only play a role during burst or violent exercise (Ristori & Laurent 1985). It is generally accepted that at a sustained cruising speed, fish use lipid metabo- lism to drive red muscle (Gordon 1968, Blaxter 1969, Bilinski 1974). However this was not reflected in the levels of the FFA which showed no significant difference with the control group at any swimming velocity, nor was there any change in the 'exhausted group'. Another characte- ristic of the 'acute stress syndrome' due to exercise is an elevation of the K+ plasma concentra- tion. This result was confirmed in a study with rainbow trout (Oncorhynchus mykiss). In a moderate exercised group (1.5 BL per sec) potassium was significantly increased from controls after 2 h of exercise until the end of the experiment after 24 h. In this study, it was hypothesized that the elevated plasma potassium concentrations after moderate exercise may be the result of an efflux of potassium from the muscle tissue caused by a blockage of the Na+/K+ ion pumps due

120 Chapter 7 to a limited production of ATP (Nielsen et al. 1994). This hypothesis is confirmed by two observations: a) the level of the increased potassium concentration is dependent on the intensity level of exercise in rainbow trout (Thomas et al. 1987); b) muscular contraction can induce an efflux of potassium ions from the myoplasma into the extracellular fluid (Sjogaard 1990). The question remains regarding the cause for the swimming failure in the exhausted group. Several mechanisms are plausible. First, the rise of the [lactate] by 860% may interfere with muscle contractility. A second key toxic event as proposed by Wood et al. (1983) is an intracellular acidosis of the intracellular compartment. With in vivo 31P-NMR the intracellular pH of muscle tissue can easily be determined based on the chemical shift between creatinephosphate (PCr) and Inorganic Phosphate (van den Thillart et al. 1989, van Ginneken et al. 1995, 1996, 1999). In combined studies using a Blazka respirometer and a 31P-NMR spectrometer, carp and trout were exposed to severe exercise in a swim-tunnel. The dynamics of the recovery process of energy rich compounds and intracellular pH were followed with in vivo 31P-NMR while the response of energy rich compounds were determined in different body compartments (blood, white-, red muscle and liver) via conventional biochemical methods. In this study, we observed a dichotomous response of trout and carp to exhaustive exercise: 50% of the animals recovered while 50% died. Comparing the data of the survival groups with the mortality groups for intracellular pH and depletion of the energy stores revealed that there was no significant difference for both fish species between survivors and non-survivors for intracellular pH which dropped to a value around 6.6-6.7. However, the difference between the survivors and non-survivors in depletion of the energy stores was tremendous resulting in a near depletion of the PCr and ATP pool in the non- survivors. Based on these observations, we concluded that the cause for fish mortality after extreme exercise is a depletion of the energy stores and not an internal acidosis of the muscle compartment (van Ginneken et al. 2001). In literature, limited data on swimming performance of eels or other anguilliform swimming teleost are available (Webb, 1975; McCleave 1980, Müller et al. 1995). From the scarce field data the optimal swimming speed of eel can be calculated. Ellerby et al. (2001) assumed a 60 cm eel with a speed of 0.48 BL/s (=0.29 m/s), at a constant swimming speed, would perform the distance from the European coast to the Sargassosea (approximately 5000 km) in 28.5 weeks. This swimming speed is extremely low for fish because burst activity i.e. for salmon may correspond to 10-11 BL/sec (Videler 1993). Results of this study are also indicative that eel-like

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(anguilliform) swimming is more suitable for long term sustained swimming then for burst activity. In conclusion, this relatively low cruising speed of up to 2 BL/sec for eel could be characteristic for this catadromic long distance traveler.

Perspectives: Because Ellerby et al. (2001) demonstrated a difference in muscle performance and properties between yellow or silver (=migratory stage) eel stages, this study with a Blazka swim tunnel and yellow eel has to be extended in future studies with similar experiments with silver eel

ACKNOWLEDGMENTS We thank Ir.J. van Rijsingen and Drs. Carolien Vancoillie, Royaal BV, Helmond, the Netherlands, for providing the yellow eel. Vincent van Ginneken is supported by a grant of the Foundation for Technical Research (STW), which is subsidized by the Netherlands Organization for Scientific Research (NWO), STW-project no. LBI66.4199.

REFERENCES

Balm, P.H.M. & T.G. Pottinger, 1993. Acclimation of Rainbow Trout (Oncorhynchus mykiss) to low environmental pH does not involve an activation of the pituitary-interrenal axis, but evokes adjustments in branchial structure. Can.J.Fish.Aquat.Sci. 50: 2532-2541. Balm, P.H.M., P. Pepels, S. Helfrich, M.L.L. Hovens, & S.E. Wendelaar Bonga, 1994. Adrenocorticotropic hormone (ACTH) in relation to interrenal function during stress in tilapia (Oreochromis mossambicus). General Comparative Endocrinology 96: 447-460. Bilinski, E., 1974. Biochemical aspects of fish swimming. In: D.Mallins and J.Sargent (Eds.), Biochemical perspectives in Marine Biology, Vol. 1, p.239-288, Academic Press, New York. Blaxter, J.H.S., 1969. Swimming speeds of fish. FAO (Food and Agricultural Organization, United Nations) Fish.Rep. 62: 69-100. Brett, J.R., 1965. The swimming energetics of salmon. Scientific American 213: 80-85. Brett, J.R., 1967. Swimming performance of sockeye salmon (Oncorhynchus nerka) in relation to fatigue time and temperature. J.Fish.Res.Bd.Can. 24: 1731-1741. Brett, J.R., 1973. Energy expenditure of Sockeye Salmon Oncorhynchus nerka, during sustained performance. J.Fish.Res.Board. Can. 30: 1799-1809. Brett, J.R. & N.R. Glass, 1973. Metabolic rate and critical swimming speeds of sockeye salmon (Oncorhynchus nerka) in relation to size and temperature. J.Fish.Res.Board Can. 3: 379- 387. Christiansen, J.S. & M. Jobling, 1990. The behavior and relationship between food intake and growth of juvenile Arctic charr, Salvelinus alpinus L., subjected to sustained exercise.

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Can.J.Zool. 68: 2185-2191. Ellerby, D.J., I.L.Y.Spierts & J.D.Altringham, 2001. Slow muscle power output of yellow- and silver-phase European eels (Anguilla anguilla L.): changes in muscle performance prior to migration. J.Exp.Biol. 204: 1369-1379. Gilham, I.D. & B.I.Baker, 1985. A background facilitates the response to stress in teleosts. J.Endocr.105: 99-105. Gillis, G.B., 1998. Environmental effects of undulatory locomotion in the American eel Anguilla rostrata: kinematics in water and on land. J.Exp.Biol. 201: 949-961.

Gordon, M.S., 1968. Oxygen consumption of red and white muscle from tuna fishes. Science 159: 87-90. Hunter, J.R., 1971. Sustained swimming speed of Jack Mackerel, Trachurus symmetricus. Fishery Bulletin 69: 267-271. Johnston, I.A. & T.W. Moon, 1980. Exercise training in skeletal muscle of brook trout (Salvelinus fontinalis). J.Exp.Biol. 87: 177-194. Mazeaud, M.M., F. Mazeaud & E.M. Donaldson, 1977. Primary and secondary effects of stress in fish: some new data with a general view. Trans.Am.Fish.Soc. 106: 201-212. McCleave, J.D., 1980. Swimming performance of European eel (Anguilla anguilla L.) elvers. J.Fish.Biol. 16: 445-452. Milligan CL, & C.M. Wood, 1986 Intracellular and extracellular acid-base status and H+ exchange with the environment after exhaustive exercise in the rainbow trout. J.Exp.Biol. 123: 93-121. Müller, U.K., J.H. Smit, E.J. Stamhuis, & J.J. Videler, 1995. Flow field around a swimming juvenile eel. BIONA report 10, editor W.Nachtigall, Fischer Verlag. Nakano, T. & N. Tomlinson, 1967. Catecholamine and carbohydrate metabolism in rainbow trout (Salmo gairdneri) in relation to physical disturbance. J.Fish.Res.Bd.Can. 24: 1701-1715. Nielsen, M. & L. Boesgaard, 1994. Plasma levels of lactate, potassium, glucose, cortisol, growth hormone and triiodo-L-thronine in rainbow trout (Oncorhynchus mykiss) during exercise at various levels for 24 h. Can.J.Zool. 72: 1643-1647. Priede, I.G. & F.G.T. Holliday, 1980. The use of a new tilting tunnel respirometer to investigate some aspects of metabolism and swimming activity of the plaice (Pleuronectus platessa L.) J.Exp.Biol. 85: 295-309. Ristori, M.T. & P. Laurent, 1985. Plasma catecholamines and glucose during moderate exercise in the trout: comparison with bursts of violent activity. Exp.Biol. 44: 247-253. Smit, H., 1965. Some experiments on the oxygen consumption of goldfish (Carassius auratus L.) in relation to swimming speed. Can.J.Zool. 43: 623-633. Smit, H., J.M. Amelink-Koutstaal, J. Vijverberg, & J.C. Von Vaupel-Klein, 1971. Oxygen consumption and efficiency of swimming goldfish. Comp.Biochem.Physiol. 39A: 1-28. Smith, R.J.F., 1985. The Control of Fish Migration. Editors: B.Heinrich, W.S.Hoar, K.Johansen, H.Langer, G.Neuweiler, D.J.Randall, 243 pp., Springer-Verlag, Berlin Heidelberg. Sjogaard, G., 1990. Exercise-induced muscle fatigue: the significance of potassium. Acta Physiol.Scand. 140: 5-51. Thomas, S., J. Poupin, G. Lykkeboe, & K. Johansen, 1987. Effect of graded exercise on blood gas tensions and acid-base characteristics of rainbow trout. Respir.Physiol. 68: 85-97. Van Dijk, P.L.M., G.E.E.J.M. Van den Thillart, P. Balm, & S. Wendelaar Bonga, 1993a. The influence of gradual water acidification on the acid/base status and plasma hormone levels in the carp. Journal of Fish Biology 42: 661-671. Van Dijk, P.L.M., G.E.E.J.M. Van den Thillart, & S. Wendelaar Bonga, 1993b. Is there a

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synergistic effect between steady-state exercise and water acidification in carp ? Journal of Fish Biology 42: 673-681. Van Ginneken, V., G. Van den Thillart, A. Addink, & C. Erkelens, 1995. Fish muscle energy metabolism measured during hypoxia and recovery: an in vivo 31P-NMR study. Am.J.Physiol. 268: R1178-R1187. Van Ginneken, V., G. Van den Thillart, A. Addink, & C. Erkelens, 1996. Synergistic effect of acidification and hypoxia: an in vivo 31P-NMR study in fishes. Am.J.Physiol. 271: R 1746-R1752.

Van Ginneken, V., G. Van den Thillart ., H. Muller, S. Van Deursen, M. Onderwater, J. Visee, V. Hopmans, G.Van Vliet, & K. Nicolay, 1999. Phosphorylation state of red and white muscle in tilapia during graded hypoxia: an in vivo 31P-NMR study. Am.J.Physiol. 277: R1501-R1512. Van Ginneken, V., T. Sundermeier, R. Boot, K. Coldenhoff, J. Hollander, F. Lefeber, & G. Van den Thillart (2001). The question 'why do fish die after severe exercise ? ' revisited: an in vivo 31P-NMR Study. Am.J.Physiology, submitted. Van Ginneken, V. & G. Van den Thillart, 2000. Eel fat stores are enough to reach the Sargasso. Nature 403: 156-157. Videler, J.J. 1993. Fish Swimming. Chapman & Hall, London, Fish and Fisheries Series 10, 260 pp. Virtanen, E., & L. Forsman, 1987. Physiological responses to continuous swimming in wild salmon (Salmo salar L.) parr and smolt. Fish Physiology and Biochemistry 4: 157-163. Webb, P.W. 1971. The swimming energetics of trout 11. Oxygen consumption and swimming efficiency. J.Exp.Biol. 55: 521-540. Webb, P.W. 1975. Hydrodynamics and energetics of fish propulsion. Bull.Fish. Res.Board.Can. 190: 77-105. Wendelaar Bonga, S.E., 1997. The stress response in fish. Physiological Reviews 77: 591-625. Wood, C.M., J.D. Turner, & M.S. Graham, 1983. Why do fish die after severe exercise? J.Fish.Biol. 22: 189-201.

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Eel fat stores are enough to reach the Sargasso

Vincent J.T. van Ginneken and Guido E.E.J.M. van den Thillart.

Institute of Biology Leiden,Van der Klaauw Laboratory,POB 9516,2300 RA Leiden, The Netherlands.

Published in: Nature (2000) 403: 156-157

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De generatio spontanea: paling onstaat uit het binnenste van de Aarde op plaatsen waar verrotting plaatsvindt (vergelijk passage van Aristoteles) Het gemeene seggen alhier is dat Ael en Palingh, uijt een bedervinge in stinckende poelen en slooten voort gebracht worden; Andere seggen weder dat den dauw inde maent van Meij de Palingh en ael voortbrengt. (Antoni van Leeuwenhoek, Brief No. 33 [21], 5 october 1677).

Chapter 8

Eel fat stores are enough to reach the Sargasso

It has long been assumed that the European eel (Anguilla anguilla) migrates to the Sargasso Sea - a region of the Atlantic Ocean between the Azores and the West Indies - to spawn1-3 . During the past decade, however, the number of glass eels has inexplicably dropped4, and it has been suggested that a shortage of fat stores in adults, resulting from diminished food resources for juveniles in inland waters, may prevent the starving silver eels from reaching the spawning grounds4-6. But we find that the energetic cost of the 6,000-km migration is actually quite low, with 60% of the fat store remaining available for the developing gonads. Silver eels leaving the coasts of Europe between September and November are likely to reach the Sargasso Sea from February to June3, so the average swimming speed for a female silver eel 1 metre long is about half a body-length per second. The estimated energy required is around 30% of the total energy at the start7. The fat reserves of migrating silver eels range from 10 to 28% (ref. 5), which suggests that most European silver eels cannot finish the journey.

Table 1: Energy cost of swimming of migrating silver eels. Values are mean (± s.d.) for five eels.

Mean O2 Consumption Energy Consumption Swimming Distance Energy Cost of -1 -1) -1 -1 -1 (ml O2 kg h [cal g h ] [km] Swimming [cal-1 g-1 km-1]

46.13 (±9.90) 0.222 (±0.048) 387.1 (±11.27) 0.137 (±0.026)

We therefore measured the energy consumption of silver eels (1 m long) that were swimming continuously at half a bodylength per second for 10 days (Fig. 1). We converted the oxygen- consumption data to fat oxidation by using the oxycaloric value of fat9, the only suitable fuel. The eels’ fatconsumption rates when resting and swimming were 10.11±0.36 and 23.06±0.41 mg fat per kg per hour, respectively. We can use these values to calculate the energy costs for migrating eels. The fat content of silver eels ranges from 10 to 28% with a mean of 20% (ref. 5). Assuming that a 1-m adult silver eel weighing 2 kg, of which 400 g is fat, migrates 43.2 km a day (at half a body-length per second), it will take 139 days for it to reach the Sargasso Sea. During this time it will use about 154 g fat, corresponding to 38.5% of its fat stores.

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It has been suggested10 that the energy cost is between 0.329 and 0.417 cal per g per km, some 2.4–3.0 times higher than our results of 0.137 cal per g per km (Table 1). The energy costs may actually be even lower because silver eels migrate at lower temperatures and may be using westward currents. According to our data, about 60% of the eel’s initial fat reserve can be used for gonad development at the end of the journey. Based on a mean energy content of fish eggs of 23.48 kJ per g dry weight11, a 2-kg female silver eel would be able to produce 413 g of eggs. This corresponds to a gonad–somatotrophic index of 22, which is a normal value for hormone-treated animals7.

Figure 1: Oxygen consumption of a silver eel (1.5 kg, 90 cm long), swimming at half a body- length per second, measured continuously between 90 and 80% air saturation. Adult migrating silver eel were studied in a 127-litre swim tunnel8, with a water temperature of 14 ˚C and salinity of 33‰. The swim tunnel was calibrated using a laser doppler method at the Hydraulics Laboratory TU, Delft. There was a linear relation between the revolution rate of the propeller and the water velocity, which enabled us to apply a water velocity of half a body-length per second for every eel.

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Vincent J. T. van Ginneken, Guido E. E. J. M. van den Thillart Institute of Ecological and Evolutionary Sciences, Integrative Zoology, van der Klaauw Laboratorium, PO Box 9511, 2300 RA Leiden, The Netherlands

1. J. Schmidt, Nature 111, 51-54 (1923). 2. M. Miller and J.D. McCleave, Journal of Marine Research 52, 743-772 (1994). 3. H.Fricke and R. Kaese, Naturwissenschaften 82, 32 -36(1995). 4. M. Castonguay et al., Fish. Oceanogr. 3, 197-203 (1994). 5. H. Svedäng and H. Wickström, J.Fish.Biol. 50, 475-486 (1997). 6. F.W. Tesch, The eel . Biology and Management of Anguilloid eel. (Chapman and Hall, London, 434 pp, 1977). 7. I. Boëtius and J.Boëtius, Dana 1, 1-28 (1980). 8. P.L.M. van Dijk et al., J.Fish.Biol. 42, 661-671 (1993). 9. J.M. Elliot and W. Davison, Oecologia 19, 195-201 (1975). 10. K. Schmidt-Nielsen, Science 177, 222-228 (1972). 11. R.J. Wootton, Symp.Zool.Soc.Lond. 44, 133-159 (1979).

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Eel migration to the Sargasso: remarkably high swimming efficiency and low energy costs.

Vincent van Ginneken1, Erik Antonissen1, Ulrike K Müller2, Ronald Booms3, Ep Eding3, Johan Verreth3 and Guido van den Thillart1

1) Institute of Evolutionary and Ecological Sciences, Integrative Zoology, Leiden University, POB 9511, 2300 RA Leiden, The Netherlands, E-mail: [email protected]

2) Department of Experimental Zoology, Wageningen Agricultural University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands

3) Department of Fishculture and Fisheries, Wageningen Agricultural University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands

Correspondence should be addressed to Dr.V.van Ginneken (e-mail: [email protected])

Keywords: Eel, Anguilla anguilla, trout, swimming efficiency, metabolic costs, muscle performance, swimtunnel, cost of transport.

Published in: The Journal of Experimental Biology (2005) 208:1329-1335

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De generatio spontanea: paling onstaat uit dauw in de maand Mei Tis hier te lande bij na een algemeen seggen, dat de Ael en Paling sonder voorteelinge geschapen werd, en of ik al seg, dat sulks voor mij onmogelijk is te begrijpen, en dat bij aldien sulks waar was, waar om dat als dan, niet te voorschijn komen, soo een groote menigte van Alen en Palingen dat onse water gragten, daar door als vervult werden. Dit seggen en heeft alleen geen plaats bij de Gemene Man maar het komt mij selfs van Aansienlijke en Geleerde Luijden te vooren. Ja men komt soo verre, dat men seijt te weten hoe, en waar de Ael en Paling voort geteelt werd, en die is dese. Men neemt inde maant Meij, twee sooden of spitten Aerde, die met gras bewassen sijn, en men leijt die twee sooden, met der selver gras bewassen sijden, op malkanderen, en dus legtmen deselve voor sonnen ondergang, soo verre int water, dat het gras met de superfitie of oppervlak, van het water gelijk komt. Wanneer nu des avonts den dauw wel gevallen is, soo neemt men des anderen daag’s mergens de sooden aarde uijt het water, en men vint tusschen het gras, verscheijde seer kleijne Alen, die men als dan oordeelt, dat uijt den dauw sijn voortgekomen, en tot meerder bevestinge voegtmen daar bij, dat soo het niet en dauwt, soo salmen geen jonge Alen int gras vernemen. Dit sijn dan de redenen die men weet te geven, dat de Alen door den dauw werden voort geteelt. (Antoni van Leeuwenhoek, Brief No. 123 [75], 16 september 1692).

Chapter 9

Eel migration to the Sargasso: remarkably high swimming efficiency and low energy costs

SUMMARY One of the mysteries of animal kingdom is the long-distance migration of 5,000-6,000 km of the European eel (Anguilla anguilla L.) from the coasts of Europe to its spawning grounds in the Sargasso Sea. The only evidence for the location of the spawning site of the European eel in the Sargasso Sea is the discovery by Johannes Schmidt at the beginning of the previous of the smallest eel larvae (leptocephali) century near the Sargasso Sea. For years it has been questioned whether the fasting eels have sufficient energy reserves to cover this enormous distance. We have tested Schmidt’s theory by placing eels in swim tunnels in the laboratory and allowing them to make a simulated migration of 5500 km. We find that eels swim 4 to 6 times more efficiently than non eel-like fish. Our findings are an important advance in this field because they remove a central objection to Schmidt's theory by showing that their energy reserves are, in principle, sufficient for the migration. Conclusive proof of the Sargasso Sea theory is likely to come from satellite tracking technology.

INTRODUCTION At the beginning of the previous century Johannes Schmidt found the smallest eel larvae (leptocephali) of the European eel (Anguilla anguilla L.) near the Sargasso Sea (Schmidt, 1923) and the bigger leptocephali nearer to the European coast. This is the only evidence to date that places the spawning grounds in the Sargasso Sea (neither eggs nor mature adults have ever been found in this area). For Schmidt’s theory to be substantiated, the following three conditions must be met. (1) Adult European eels must be able cover a distance of 6,000-km in a fasting state, implying that they have sufficient energy reserves to cover this enormous distance (Tucker 1959). (2) Mature European eels and fertilized eggs must be found in the Sargasso Sea. (3) Eel larvae must be shown to migrate towards the European coasts. Concerning condition (3), the most recent observations on larval migration patterns were published by McCleave et al. (1987). Several questions concerning the large variation in age (Antunes & Tesch 1997) and genetic make up (Wirth & Bernatchez 2001) of glass eels collected at different places and times (suggesting the existence of more than one spawning site) have not yet been resolved, however. To test condition (2), Tesch’s group (Post & Tesch 1982) have tried, so far without success, to catch adult eels in the Sargasso.

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Until now there have only been two reports of silver eel A. anguilla caught incidentally in the open Atlantic (Ernst 1977, Bast & Klinkhardt 1988). Concerning condition (1), Tucker (1959) expressed severe doubts as to whether the European eel would be able to swim across the ocean and suggested that all European eels are the offspring of the American eel. Tucker's 'new solution to the Atlantic eel problem' provoked a long debate (D'Ancona & Tucker 1959, Deelder & Tucker 1960) and was finally rejected when a distinction was made between the two Atlantic eel species based on allozymes (Williams & Koehn 1984), enzymes (Comparini & Rodino 1980), mitochondrial DNA (Avise et al. 1986, Avise et al. 1990, Tagliavini et al. 1995) and genomic DNA (Nieddu et al. 1988). Hence, if we assume a long- distance spawning migration to the Sargasso Sea, the energy reserves may easily be critical (Svedäng & Wickström 1997). An estimation of the energy required to cover the distance was presented in a recent paper. Based on the oxygen consumption rates during a 10-day swim trial, the equivalent fat consumption extrapolated to 6000 km was 120 g kg-1 or 40% of the initial fat reserve (van Ginneken & van den Thillart 2000). Female silver eels of 0.8-m body length (BL) swimming at 0.5 BL s-1 would cover a distance of 6000 km in about 180 days. However, direct proof that European eels are able to swim the long distances between Europe and the Sargasso Sea is still missing, and the question remains as to whether eels can continue swimming up to 180 days at the same low energetic cost. The objective of this study was to determine the energy costs of swimming over a complete 5500 km swim trial in the laboratory.

MATERIAL AND METHODS We conducted two sets of experiments, one over the entire distance of 5500 km, which lasted 173 days, and another set over the duration of 1 week. The second set served as confirmation of results obtained during the first set by comparing our measurements on eel with the performance of another well-studied fish species, trout. In experiment 1, we performed respirometry in combination with bomb-calorimetry on the eel carcasses. In experiment 2, using trout and eel, we performed respirometry only in the swim tunnels.

Animals For experiment 1, 3 years old hatchery eels (Anguilla anguilla, N=30, 860 ± 81.9 g and 73.1 ± 3.8 cm) were used. In experiment 2, we used eels (A. anguilla, N=5, 155.0 ± 18.3 g, 43.2 ± 3.2 cm) and trout (Oncorhynchus mykiss Walbaum, N=5, 161.5 ± 21.5 g, 24.6 ± 1.0 cm) of the same body weight.

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The eels used both in experiment 1 and 2 were in the non-migratory yellow stage. Eels were obtained from Royaal B.V., Helmond, The Netherlands, and trout were obtained from the Dutch Organization of Fisheries, O.V.B., Geertuidenberg, The Netherlands.

Flow tank experiments In experiment 1, the oxygen consumption rates of eels (N =9) swimming at 0.5 BL s-1 were measured over a period of 173 days in 2 m Blazka-type flow tanks with a volume of 127.1 ± 0.9 liter. In addition, to establish the routine metabolic rate (RMR) we measured, the oxygen consumption over the same period of 173 days, of six eels resting in Blazka-type flow tanks with continuous water refreshment. The cross section of the flow tanks is circular with an inner diameter of 190 mm. Outside the boundary layer the flow speed is constant over the cross section of the inner tube. The flow tanks were calibrated using a Laser Doppler method. Thus the eels swam at a constant known speed with negligible wall effect (van den Thillart et al. 2004). Swimming experiments were performed over 173 days at 12:12 hours day:night light cycle at a temperature of 19.0 ° ± 0.3 ° C at 0.5 BL s-1 The illumination in the climatized room was switched to 670 nm light (bandwidth 20 nm) during experiments. Based on pigment changes during silvering it is assumed that this far-red light is invisible for eels (Pankhorst and Lythgoe, 1983). The time lapse between the start of migration and the first leptocephali in the Sargasso Sea is about 6 months. Thus to cover a distance of 5000-6000 km in 6 months requires a mean swimming speed of 0.4 m.s-1 (Ellerby et al. 2001). For experiment 2, five eels and five trout were placed in the same 127 l flow tanks at a constant temperature of 18 ° ± 0.3 ° C under the same illumination protocol. Eels swam at 0.5 BL s-1 (21.5 ± 1.6 cm/sec), whereas the trout swam at a slightly higher speed of 0.7 BL s-1 (17.2 ± 0.7 cm/sec). We ensured that both species swim at their maximum range or optimal swimming speed, which is the relevant speed for migration, to facilitate the comparison between the eel’s and the trout’s costs of transport. The optimal swimming speed of trout is available in the literature (Webb, 1971), whereas the value for eel is based on a study of eel muscle efficiency (Ellerby et al. 2001) (for a more detailed explanation, see Discussion). Swimming behavior was recorded at regular intervals during the entire period with an infrared video camera (frame rate 25 Hz) during the dark period to document the position of the animal in the flow tank as well as to detect possible long-term changes in swimming behavior.

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Before sampling the animals they were quickly anaesthetized with 300 p.p.m. MS222 (3- aminobenzoic-acid-ethyl-ester methane sulfonate salt; Sigma, St. Louis, USA). All experiments were approved by the local committee on animal experimentation.

Oxygen consumption The oxygen level in the tunnel was measured continuously using an oxygen electrode (Mettler Toledo, Tiel). The oxygen consumption rate was calculated from the oxygen decline after automatic closure of the water-inlet by a magnetic valve. The oxygen levels changed between 85 and 75% air saturation. The valve was normally open allowing a refreshment rate of 5-7 l.min-1 and automatically operated between 14:00 and 17:00 h to measure oxygen consumption. The oxygen value was not allowed to fall below 75% in order to prevent the animals from becoming hypoxic (van den Thillart and van Waarde, 1985). -1 From the decrease in the O2-concentration, the rate of oxygen consumption (VO2 in mg O2 h -1 -1 -1 kg ) was calculated from the formula: VO2 = 127.δ[O2].δ t , where: δ[O2].δ t is the decrease of the oxygen content per hour. Oxygen consumption data were corrected for the decline in mass of the animals.

Carcass analyses To quantify the energy cost of transportation by a second method, independently from our respiratory measurements, we analyzed the changes in body composition. Carcass analyses were performed according to ISO-standards (International Organization for Standardization, Animal feeding stuffs; ISO 5983 and ISO/DIS 6492; Geneva, Switzerland). After weighing, fish samples were cut into pieces of about 3 cm and nearly submerged in water in a glass beaker. The samples were autoclaved at 2 atm (2.013 x 105 Pa) at 120o for 4 hours. They were then homogenized and subsequently sampled in triplicate for dry matter, protein and fat analyses. Dry matter content was measured by freeze drying of the sub samples to constant mass. Protein was measured according to procedures desribed in ISO 5983 (1979). For the fat determination, freeze dried sub-samples were extracted as described in ISO/DIS 6492 (1996).

Calculations and statistics In order to calculate the cost of transportation (COT: mean gross energy costs of transportation; Schmidt-Nielsen, 1972), total energy consumption was first calculated from oxygen consumption by multiplying the mean measured oxygen consumption

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-1 -1 (Table 1, in ml O2 kg fish h ) with the number of swimming hours (4152 hours is 173 days) -1 and the applied energy conversion factor for respirometry of 18.89 kJ ml O2 (Elliot & Davison 1975). This gives a total energy consumption of 2316.58 kJ kg-1 fish over 5533.2 km or a COT of 0.42 kJ.kg-1.km-1. Alternatively the energy used during the 5533 km run was calculated by bomb-calorimetry. Bomb-calorimetry could be only used once on eels at the start of the experiment, so a control group of 15 animals was also measured for comparison with the swim- and the rest-groups (Table 2).

The calculations for the bomb-calorimetry were as follows: 1. Initial wet body mass (g) * initial dry matter fraction (%) = initial dry matter content (g dry mass fish-1) 2. End wet body mass (g) * end dry matter fraction (%) = end dry matter content (g dry mass fish-1) 3. Initial energy content (kJ g-1 dry mass) * initial dry matter content (g dry mass fish-1) = total energy content begin (kJ fish –1). 4. End energy content (kJ g-1 dry mass) * end dry matter content (g dry mass fish-1) = total energy content end (kJ fish –1). 5. total energy content begin (kJ fish –1) - total energy content end (kJ fish –1) = total energy difference (kJ fish –1). 6. Total energy difference (kJ fish –1) / geometric bodyweight (g) * 1000 = total energy usage (kJ kg-1).

With: geometric bodyweight = EXP (LN start wet body weight + LN end wet body weight)/2

The COT was total energy usage (= total energy consumption in kJ kg-1 fish, Table 1) multiplied by the applied energy conversion factor for bomb-calorimetry of 33.73 kJ/g dry mass (Brafield and Llewellyn 1982). Values are means ± S.D. Statistics were performed using a two way independent sample t-test using SPSS 10.0. (*** P≤ 0.001, ** P≤0.05, * P≤0.01).

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Oxygen consumption Bomb-calorimetry Swimgroup Restgroup Swimgroup Restgroup (n=9) (n=6) (n=9) (n=15) Animal length 74.7 70.6 74.7 71.7 (cm) ± 3.4 ± 3.6 ± 3.4 ± 3.3 Wet weight begin 914.7 795.0 *** (g) ± 58.4 ± 71.9 Wet weight end 734.3 691.1 (g) ± 44.9 ± 70.7 Wet weight loss 180.3 103.9 *** (g) ± 38.2 ± 26.3 Dry matter loss 84.3 42.7 *** (grams kg-1 fish) ± 12.6 ± 22.2 Oxygen consumption 29.55 *** 14.26 *** (ml 02 kg-1 fish h-1) ± 4.2 ± 1.8 Total energy consumption 2316.58 1122.86 *** 3450.6 1941.0 *** (kJ kg-1 fish) ± 221.31 ± 107.72 ± 723.9 ± 1172.0 COT (kJ /kg /km) 0.42 0.62

Table1: Energy consumption parameters of female yellow eels after 6 months of rest and after 6 months of swimming at 0.5 BL s-1. The eels swam 5533 ± 354 km over 173 days. For details of the methods used, see text. COT: mean gross Energy Costs of Transportation (Schmidt-Nielsen 1972). * : P≤0.05 denotes significant difference ; ** : P≤0.01 denotes significant difference ; *** : P≤0.001 denotes significant difference.

Start (N=15) 5500 km Swim (N=9) 6 months Rest (N=15) Fat 67.92 (1.91) 68.16 (2.47) 68.09 (2.20) Protein 28.17 (1.79) 28.28 (2.16) 27.99 (1.90) Carbohydrate 0.9 (0.43) 0.57 (0.49) 0.87 (0.53) Ash 2.97 2.99 3.05 Dry matter 49.57 (2.41) 50.25 (2.86) 50.77 (2.18)

Table 2: Body-constitution in % of dry mass of female yellow eels at the start and after 6 months swimming or resting.

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Figure 1: Oxygen consumption of fasting yellow eels from a hatchery (860 ± 81.9 g, 73.1 ± 3.8 cm BL) during a 6 months period of rest (circles) or 6 months of continuously swimming at 0.5 BL s-1 (diamonds) at 19 ° C. Regression lines: Rest-group: Y=0.0326 X + 25.294; Swim- group: Y=0.0394 X + 54.86.

RESULTS The 'swimming' eels in experiment 1 used 2.1 times more oxygen than the 'resting' eels -1 -1 (29.55 vs. 14.26 ml O2 .kg h , Table 1). The regression lines show that the oxygen consumption of the swimming eels increased by 2.56% over nearly 6 months, that of the resting group increased by 2.66% (Fig. 1). There was a significant difference in mass loss: 13.1% in the resting group and 19.7% in the swimmers. Analysis of body constituents of the eels at the start and end of the experiment revealed that the ratio of all three substrates (lipid, carbohydrate, and protein) remained constant despite significant mass losses (Table 2). This means that body composition did not change during the six months and that fat, protein, and carbohydrate were metabolized in the same proportion. A similar result was also found in reef fish migrating over much smaller distances (Stobutzki, 1997). We obtained two independent estimates for cost of transport. From the first method, oxygen uptake, (using the oxycaloric value of the three substrates (Elliot & Davison 1975), we obtained a value of 2317 kJ kg-1 fish for the energy cost of 6 months swimming covering a distance of 5500 km. This corresponds to a COT value of 0.42 kJ kg-1 km-1. The swimming group lost more

136 Chapter 9 total body mass than the resting group (180.3 g compared with 103.9 g; Table 1). Hence, based on the second method, which uses mass loss, body composition and energy conversion factors (Brafield and Llewellyn 1982), we calculated that energy used for swimming was 3450 kJ kg-1 fish, corresponding to a COT value of 0.62 kJ kg-1 km-1. The two COT estimates obtained independently (Table 1) are of the same order of magnitude. In experiment 2, where eels and trout swam in identical experimental set ups, we measured an oxygen consumption of respectively 43.9 ± 8.42 mg O2 kg-1 h-1 and 130.4 ± 9.49 mg O2 kg-1 h-1. During 7 days the eels and trout covered a mean distance of 132.5 ± 12.1 km and 102.8 ± 2.3 km, respectively. From these data we calculated COT values for eel and trout of 0.68 and 2.73 kJ kg-1 km-1 respectively. Our video recordings of the fish swimming in the flow tank show that the fish were swimming in the free-stream and did not benefit from wall effects: the fish swim mostly in the centre of the flow tank and therefore are more than 2 (eel) until 3 (trout) tail heights removed from the wall (results not shown).

DISCUSSION Efficiency, commonly defined as the rate of useful energy expenditure divided by the total rate of energy consumption, is a crucial parameter for animals migrating over long distances. During long-distance migration, animals are likely to maximize the distance covered per given fuel unit, which corresponds to maximizing efficiency. Amongst the various eel species, the European eel need to migrate the farthest to reach its spawning grounds: European eel (A. anguilla) 5500 km (Schmidt 1923), American eel (A. rostrata) 4000 km (Tucker 1959, McCleave et al. 1987); Australian eel (A. australis) 5000 km (Jellyman 1987) and Japanese eel (A. japonica) 4000 km (Tsukamoto 1992). So European eels need to be very efficient swimmers. There are various levels of energy conversion in a swimming animal. The overall metabolic efficiency (how much heat is generated at a given swimming speed) comprises the efficiencies of various processes, e.g. propeller efficiency (how much momentum is gained by the animal and wasted in the wake) and the muscle efficiency (how many ATP molecules are used per myosin-head cycle). The concept of efficiency used here corresponds to overall metabolic efficiency and encompasses e.g. propeller and muscle efficiency for example. During locomotion, propeller and muscle efficiency are likely to contribute significantly to overall metabolic efficiency, so it is interesting to compare the hydrodynamic and muscle performance of eel to those of other undulatory swimmers.

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To reduce costs of transport and increase overall metabolic efficiency, all or some of the processes that determine the costs of transport can be optimized. Efficiency can be improved most effectively by improving those processes in which efficiency increases nonlinearly and progressively with a given performance parameter. Performance parameters that only weakly or linearly affect efficiency are less suitable to bring about a drastic increase in efficiency. In order to explain the remarkable difference in cost of transport between eel and trout, it is important to identify the processes that cause it. To this end, we have studied the literature on propeller efficiency and muscle efficiency in undulatory swimmers.

Hydrodynamic performance and propeller efficiency A fish can alter its propeller efficiency by changing its structural design and its motion pattern. Both carangiform and anguilliform swimmers undulate their body, the former with a narrower amplitude envelope than the latter. How the shape of the body undulations affects locomotory efficiency has been estimated using analytical approximations. Lighthill’s elongated body theory (EBT) concludes that efficient swimmers should undulate only the most posterior section of their body - in the ideal case only their trailing edge - to maximise propeller efficiency (Lighthill 1971, Tytell and Lauder 2004). Daniel’s (1991) predictions differ in part: propeller efficiency of undulatory swimming decreases linearly as the rearward speed of the body wave increases relative to the swimming speed, and it is independent of the frequency and the amplitude of the body wave. Given that the swimming kinematics of trout and eel mainly differ in the amplitude envelope of their body wave, but have a similar range of body wave speeds (for a review see Videler), it is unlikely that kinematic differences between trout and eel can explain the difference in their overall metabolic efficiency. The combined effect of propeller shape and motion on performance can be studied by visualising the flow generated by anguilliform and carangiform swimmers. The ratio of forward to total momentum of the entire wake provides the mean propeller efficiency over a complete tail beat. This approach, whether using experimental or computational flow fields, requires the quantification of the three-dimensional flow in the complete wake, which so far has not been done. The currently available two-dimensional slices through the wake suggest that eels generate considerable lateral momentum, which does not contribute to the forward motion and therefore reduces efficiency (Müller et al. 2001, Tytell and Lauder 2004). Tytell estimated a hydrodynamic efficiency of 0.5 to possibly up to 0.87 (Tytell and Lauder 2004). Equivalent estimates for carangiform fish are reported in the range from 0.74 to 0.97

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(Drucker and Lauder 2001, Müller et al. 2001, Nauen and Lauder 2002a,b). These values suggest that trout has a higher propeller efficiency than eel, which does not to explain the higher overall metabolic efficiency of eels. Efficiency is also inversely related to thrust (Lighthill 1971, Daniel 1991). However, a 25% difference in swimming speed is insufficient to explain a fourfold difference in efficiency. So, the currently existing evidence on the hydrodynamics of undulatory swimming contradicts rather than explains the high swimming efficiency of eels.

Muscle performance and efficiency The efficiency with which a muscle converts chemical energy into mechanical work is important in prolonged aerobic locomotion, such as migration. Cruising is characterized by cyclic contractions at a well-defined frequency. Swimming speed depends linearly on tail beat frequency, and tail beat frequency corresponds to contraction frequency. The mechanical efficiency of muscle contractions depends on contraction speed in a non-linear fashion. This relationship can be predicted from Hill’s model of muscle contractions (McMahon 1984) and has also been documented in fish swimming muscles (Curtin and Woledge, 1993). There is a narrow range of contraction frequencies over which efficiency remains high. At contraction frequencies above and below this range, efficiency drops off progressively (McMahon 1984, Curtin and Woledge 1993). McMahon calculations (McMahon, 1984) show that maximum efficiency occurs at a contraction speed at 13% of the maximum contraction speed of the muscle, which is slightly lower speed than the speed at maximum power. To swim at maximum muscle efficiency, the fish should maintain a tail-beat frequency that allows the muscle to contract at this optimal speed. If we take the contraction frequency that maximizes power as a first approximation of the contraction speed that maximizes efficiency, we can compare eel aerobic swimming muscles to those of trout. Eel muscles deliver peak power at much lower contraction frequencies (0.5 to 0.8 Hz in silver eel; measured at 14° C; Ellerby et al. 2001) than the muscles of trout (2 to 3 Hz, measured at 11° C; Hammond et al. 1998) The swimming speeds that correspond to these contraction frequencies are 0.5 L s-1 for eel and 0.4 to 1.0 L s-1 for trout (Webb 1971). These values confirm that in our experiments both eel and trout were swimming at close to their optimal swimming, and hence the much higher COT of trout is probably not due to the trout having been forced to swim under considerably suboptimal conditions for its swimming muscles.

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Muscle fibre type recruitment and swimming speed At the low speeds used in this study, the eels will recruit only the posterior red muscle to swim continuously. As demonstrated in the work of Gillis (1998), muscle fiber type recruitment was clearly dependent upon swimming speed. A pattern of ‘posterior-to-anterior’ recruitment within a fiber type was observed as eels increased their swimming speed (figs 2,3A,F in Gillis, 1998). For example, eels typically used mainly posteriorly located red muscle (at 0.75 and 0.6 BL) to power slow-speed swimming, but would then additionally recruit more anteriorly located red muscle (at 0.45 and 0.3 BL) to swim at the higher speeds (figs2,3A in Gillis 1998). These unusual muscle activation pattern and kinematics may explain the low COT in eels compared with trout, in which most of the red muscle on each side of the body is stimulated during a tail-beat cycle-assuming that the European and American eels are similar in this regard. In contradiction to this theory/hypothesis of Gillis (1998) to explain the low swimming efficiency of eel by recruitment patterns of muscle, Wardle et al. (1995) showed that the muscle activity pattern (% time active during one tail-beat cycle) does not differ substantially between different undulatory swimmers. Wardle’s values for eel (based on Williams et al. 1989) agree with those mentioned by Gillis (1998). Compared with trout and other fish, recruitment in eel is certainly not less by a factor of 2-4. Hence it is not likely that more posterior muscle recruitment in eel can explain the many-fold difference in efficiency between eel and trout.

Metabolism and non-locomotory influences on costs of transport Overall metabolic efficiency is also influenced by the efficiency of the respiration and energy-conversion processes themselves. The whole-organism locomotory performance is determined by its metabolic machinery, bringing us to the whole-body oxygen consumption (Routine Metabolic Rate=RMR) of the animal. In this study, we found a RMR of 29.55 ± 4.2 -1 -1 -1 -1 ml O2 kg h , which corresponds to 42.21. ± 6.0 mg O2 kg h . This value is similar to values reported in literature: 35 mg kg-1 h-1 (for the similar-sized animals at 18° C, Degani et al. 1989; McKenzie et al. 2000) for eel and also the RMR measurements of other fish species (Winberg 1956). Hence, we may conclude that, based on metabolic rate comparisons with other fish species, the mitochondrial capacity remains the same. However, in the wild, eel do not migrate at the surface but in the deep sea: a migrating eels has been photographed at the Bahamas at a depth of 2,000 m (Robins et al. 1979).

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There, they experience considerably larger pressures, which might further increase metabolic efficiency at the mitochondrial level by increasing the efficiency of their oxidative phosphorylation (Theron et al. 2000). In a laboratory study in which eels were exposed eels for 21 days at 10.1 MPa hydrostatic pressure, Theron et al. (2000) demonstrated that the ADP/O ratios, calculated from mitochondrial respiration measurements, were significantly increased. Eels actually performing the migration will not only experience higher pressures, but also lower temperatures, which will also affect their efficiency. Furthermore, eels might adapt their migratory route to take advantage of favorable sea currents, which would further reduce the energy requirements. However, with the migratory routes unknown, nothing can be said about the possible energy savings from pressure, temperature and sea-current effects.

Metabolic costs and efficiency compared Our respiratory measurements and the carcass analyses suggest that eels have a much higher metabolic efficiency than trout. In eel, the COT values obtained from oxygen consumption data and carcass analyses are 0.42 and 0.62 kJ kg-1 km-1, respectively, whereas trout has much higher COT values (respirometry only) of 2.73 kJ kg-1 km-1. The COT in trout matches the value measured by Webb (1971), and is similar to other salmonids (Brett 1973) and many adult fish species (for a review see Videler 1993). This means that eel swim 4 to 6 times more efficiently than many other fish species, even across swimming styles.

In conclusion, we demonstrated in this study that fasting European eel are able to swim 5500 km, a distance corresponding to their supposed spawning area in the Sargasso Sea, with a remarkably high swimming efficiency and at low energy costs. At this moment, this high efficiency can be explained neither by propeller nor muscle efficiency. How the extremely low optimal contraction frequency of eel aerobic muscle affects muscle efficiency has not yet been studied. So far, the only evidence that eels have a remarkably high swimming efficiency comes from metabolic energy costs. The source of the eel’s remarkably high efficiency remains at present unknown, providing ample stimulation for biomechanists and physiologists alike to investigate eel migratory swimming performance.

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ACKNOWLEDGEMENTS We thank Prof. Dr. K. Dickson (California State University, Fullerton, USA), Prof. Dr. R. Blickhan (Friedrich Schiller Universität, Jena, Germany), Dr. E.D. Tytell (Harvard University, USA), Dr. I.L.Y. Spierts (Wageningen University, The Netherlands) and Prof. M. Richardson (Leiden University, The Netherlands) for helpful suggestions and improving the manuscript. This work was supported by the Foundation for Technical Research (LBI.4199), which is subsidized by the Netherlands Organization for Scientific Research (N.W.O.) and the European Commission, project (EELREP, Q5RS-2001-01836).

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Hematology patterns of migrating European eels and the role of EVEX virus

V.van Ginneken1*, B.Ballieux2, R.Willemze2, K.Coldenhoff1, E.Lentjes2, 1 3 1 E. Antonissen , O.Haenen , G.van den Thillart

1: Institute Biology Leiden (IBL), Leiden University, The Netherlands. 2: Leiden University Medical Center, Hematology/CKCL-Laboratory, Leiden University, the Netherlands. 3: Central Institute for Animal Disease Control (CIDC-Lelystad), Lelystad, the Netherlands.

* : To whom correspondence should be addressed: Dr.V.van Ginneken (e-mail: [email protected])

Key-words: European eel, Anguilla anguilla, Virus, Simulated migration, EVEX, Blood chemistry, Lactate dehydrogenase, Total Protein, Aspartate aminotransferase.

Published in : Comparative Biochemistry and Physiology Part C (2005) 140 : 97-102

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De opvatting dat de paling levend barend (vivieparie) is: kleine wormpjes in de darmen van paling zijn kleine palingen Ick heb inde maent Julij laestleden, inden darm beneden de maegh off Rob, van Palingh en Ael ontdeckt, wormkens, die een weijnich bewegingh hadden, en ontrent van dickte waren als een groff Hair, en na mijn oordeel, ontrent twintich mael soo langh, als dick; Dese wormkens hadden in haer lichaem, een doorgaenden darm, in welcke darm omtrent het Hooft, een continuele beweginge geschiede, als off het adem hale van de diertgens was; doch na de staert was de beweginge grooter, niet inden darm, maer door het gantsche lichaem; en al wat hier starck op en neer bewogen wiert, dat waren veel kleijnder Aeltgens, off wormkens, waer van ick veele door de Huijt van het Eerste aeltge sagh leven, en bewegen. Ick heb de eerst geseijde wormkens, off Aelkens, uijt de darm van Ael en Palingh genomen, ende deselvige voor mijn gesicht ontstucken gesneden, en met verwonderingh gesien, een getal van meer (na mijn gesicht oordelende) dan 200 wormkens, off Aelkens, uijt ijder vande eerst geseijde wormkens sien comen, en dit boven veel deeltgens die ick voor Eijeren, off onvolmaeckte dierkens aensach; Dit siende nam ick in gedachten, dat Ael, en Palingh, weder Ael en Palingh voortbrachten. (Antoni van Leeuwenhoek, Brief No. 33 [21], 5 october 1677). Chapter 10

Hematology patterns of migrating European eels and the role of EVEX virus.

Abstract: We show that European eels infected with the rhabovirus EVEX (Eel Virus European X)- virus, developed hemorrhage and anemia during simulated migration in large swim tunnels, and died after 1,000-1,500 km. In contrast, virus-negative animals swam 5,500 km, the estimated distance to the spawning ground of the European eel in the Sargasso Sea. Virus- positive eels showed a decline in hematocrit, which was related to the swim distance. Virus- negative eels showed a slightly increased hematocrit. Observed changes in plasma lactate dehydrogenase (LDH), Total Protein and aspartate aminotransferase (AAT) are indicative of a serious viral infection. Based on these observations, we conclude that eel virus infections may adversely affect the spawning migration of eels, and could be a contributing factor to the worldwide decline of eel.

Introduction: Worldwide, eel populations have been dwindling over the last decade. Steep declines of 90- 99% have been reported for European eel (Anguilla anguilla), Japanese eel (A. japonica), and American eel (A. rostrata) (Stone 2003). Eels are very vulnerable to environmental factors because of their complex life cycle. As a catadromic fish species, they migrate several thousand kilometers to their spawning areas. Possible adverse effects on the adults include contamination with PCBs - which are released from fat stores during their long-distance migration (Castonguay et al. 1994) - and infection with the parasitic swim bladder nematode Anguillicola crassus (Haenen et al. 1994). Furthermore, diminished fat stores due to insufficient food supplies in the inland waters (Svëdang and Wickström 1997), blockage of migration routes by power stations & power plants, and over-fishing, are all possible causes (Castonguay et al. 1994). Changes in oceanographic currents may interfere with transport of eel larvae to the European coast, and this too may contribute to the decline in eel populations (Knights 2003). However, no conclusive evidence on any of these causes has been presented yet (Dekker 2004).

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A factor, that has not received much attention to date, is the world-wide occurrence of eel viruses (van Ginneken et al. 2004). Viruses are known to affect blood-forming tissues in fish, and typically become virulent during stress (Wolf, 1988). In salmon for example, Infectious Haematopoietic Necrosis Virus (IHNV) and Viral Haemorrhagical Septicemia Virus (VHSV), both rhabdoviruses, can affect haematopoietic tissues, leading to severe anemia (Wolf 1988). The most prominent cases of rhabdovirus infections in eel populations, described in literature, are infections with EVA (Eel-Virus-America) and EVEX (Eel-Virus- European-X ). Both viruses are serologically related (Kobayashi & Miyazaki 1996). EVA was first discovered in Japan in 1974, in a shipment of American elvers, which had been stocked in Cuba (Wolf, 1988). Another virus, which was isolated in a shipment from France to Tokyo, was named EVEX because of its European origin (Sano et al., 1977). So EVEX was described for the first time in 1977, in the period when the European eel populations started to decline. At this moment it is not known if EVEX is a virus endemic to the European eel population or that it substantially spread over the past 50 years due to aquaculture practices. EVEX-virus has recently been observed in several countries worldwide (van Ginneken et al. 2004) in European eel (A. anguilla) in the Netherlands, Italy and Morocco, but also in New Zealand longfin eel (A. dieffenbachi). In this respect it is worrying that also Herpesvirus anguillae is isolated and identified in eel populations all over the world. In cultured eel in Taiwan (Ueno et al. 1992, 1996 Chang et al. 2002), in cultured eels in the Netherlands (van Nieuwstadt et al. 2001, Davidse et al. 1999, van Ginneken et al. 2004) but also (this study) in adult European eels from Lake Grevelingen. In the comprehensive study of Jørgensen et al. (1994) elvers and eel of A. anguilla were sampled on 306 occasions in Denmark, United Kingdom, France and Sweden several eel viruses were isolated like EVEX, EVA, IPN, and herpes like viruses. This study also supports our view that viruses are widespread in the eel population. For eels, long-term migration can certainly be considered a major stressful event. Therefore, one may assume that an outbreak of a virus infection in infected individuals can take place during this journey. Based on the work of Schmidt, who caught leptocephali (the larvae of the eel) in the ocean, it is assumed that the spawning grounds of the European eel are 6,000-km removed from the European continent in the Sargasso Sea (Schmidt 1923, Miller & McCleave 1994). It is generally assumed that the silver eel does not feed during its journey to the spawning grounds, which it reaches 4 to 6 months later (Tesch 1977, Fricke & Kaese 1995). In order to test this hypothesis we simulated the 5,500-km journey to the Sargasso Sea in large Blazka swim-tunnels of 127 l, comparing virus-positive and –negative European eels.

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Material and Methods

Rationale of the experiment, selection of the animals: It was initially our intention to simulate the 5,500-km migration of European eel (Anguilla anguilla L., Anguillidae, Teleostei) to the Sargasso Sea in 22 Blazka swimtunnels. We used silver eel (1500 g ± 85 cm), which where caught in the Grevelingen, (The Netherlands) during their seaward migration in September 2000. Fish were kept in sea water (33 ppt) for one month before use in the experiment. The recirculation system and swim tunnels were placed in a climatized room with a constant temperature of 15 °C. The water temperature was kept at 14 °C. Animals were kept under constant dark conditions. Of these animals, four, five and four animals stopped with swimming after approximately 500, 1,000 and 1,500 km, respectively (Figure 1). The animals were sampled live, and blood was collected while organs were investigated for virus infections. All animals were infected with the EVEX-virus (Eel-Virus-European-X (unknown). This group was called the Virus-positive group. Of the swimgroup only the 1000 (N=5) and 1500 km sample (N=4) were used for blood analysis (see Tables 1 and 2). In a second trial, to simulate the 5,500-km migration of European eel to the Sargasso Sea, we used hatchery animals (700-900 g, ± 75 cm). The experiments with virus-negative eels from a hatchery were performed in freshwater at a temperature of 19 ° C. The animals were sampled alive after 6 months and blood was collected while organs were investigated for virus infections. All animals were virus free. This group was called the Virus-negative group. Blood plasma of all animals was investigated afterwards for blood chemistry at the CKCL-laboratory of the Academic Hospital, Leiden University, The Netherlands.

Blazka swim-tunnel: The Blazka swim-tunnel has a length of 200 cm, with a diameter of the outer swim- tunnel tube of 28.8 cm and a diameter of the inner swim-tunnel tube of 19.0 cm. The volume was 127.14 ± 0.90 liter (n=5). It was calibrated with a Laser Doppler technique at the Delft Hydraulics Laboratory, Technical University Delft. The experimental set-up is described elsewhere (van den Thillart et al. 2004). The swimming speed of the water in the swim tunnels was set at 0.5 body length per second.

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Figure 1: A: Hematocrit of European eels, which were found to be infected with EVEX-virus. Eels swam for up to one month September 2000, in large 127-liter Blazka swim tunnels. The decrease of red blood cells was negatively correlated with the covered distance. ***) Denotes significantly different P≤ 0.001. Control: initially sampled; Rest: one month rest; (Swimming=shaded area): one month swimming. B: Hematocrit of virus-negative eels which swam 5,500-km during the period March-September at a continuously speed of 0.5 Body-length per second in large 127 liter Blazka swim tunnels, (swimming=shaded area, 6 months swimming). The hematocrit increased during the 6 months swim period with 11.3 % ± 4.68. (N=9). Start: initial sample in March 2001, After 5,500-km: end sample in September 2001 taken from the same animals.

Experimental protocol: The virus-positive group consisted of one Swim Group (N=13), one Rest Group (N=13), and one Initial group (N=10). The Swim group was put in Blazka swim tunnels. The Initial and Rest group were kept in flow boxes (Overtoom BV.) of 40 l connected to the same water recirculation system and sampled at the start of the experiment as a zero sample. Both Swim- and Rest- groups were kept at the same water quality conditions in the tunnels during the experiment.

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The virus-negative group consisted of one Swim Group (N= 9), one Rest Group (N= 12), and one Initial group (N= 9). All animals from virus-positive and virus-negative groups were healthy at the start of the experiment. The presence or absence of viruses in all animals was determined after sampling and dissection of the organs. The animals were not fed during the entire experimental period.

Sampling and virus-isolation: As soon as the animals stopped swimming, they were quickly anaesthetized with 300 ppm MS222 (3-aminobenzoic-acid-ethyl-ester methanesulfonate salt; Sigma, St. Louis, USA). After 3 minutes, the anaesthetized fish were taken out of the swim-tunnel and blood was collected with a heparinized syringe (flushed with 3,000 units heparin per milliliter blood). The spleen, gills, kidney and liver were removed for virus isolation by the Fish Diseases Laboratory (CIDC-Lelystad), and stored on dry ice. Samples of organs were homogenized with sterile sand in sterile medium, and tested on three different cell-lines: RTG-2 - rainbow trout gonad cells; FHM - fat head minnow cells; and EK-1 - eel kidney cells, at 15°C, 20°C, and 26°C respectively. In the case of virus infections, the infected cell line was inspected by electron microscopy followed by either immunofluorescence or immunoperoxidase methods in order to identify the virus-type (Wolf, 1988).

Analytical methods for blood samples: Blood was centrifuged at 8000 x g for 5 min. The plasma was aliquoted and stored at -80oC pending analysis at the CKCL-laboratory of the Academic Hospital, Leiden University, The Netherlands. Lactate dehydrogenase (LDH; EC 1.1.1.27), total protein and aspartate aminotransferase (AAT; EC 2.6.1.1) were measured using a Hitachi 747 analyzer (Roche, Almere). LDH was measured according to the method of the German Society for Clinical Chemistry (1972); aspartate aminotransferase according to IFCC method without pyridoxal phosphate (Klauke et al. 1993); total protein was based on the Biuret method (Camara et al. 1991). Hematocrit values were measured in 9 μl whole blood samples using an haematocrit micro-centrifuge (Bayer, Germany).

Clinical significance of blood chemistry parameters: We selected a set of parameters, which are used in human pathology as a diagnostic tool for virus infection (Burtis et al. 1996).

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The AAT activity was determined to estimate the liver function. Furthermore, we used two parameters as indicators for liver disorder and/or general catabolism: a) total plasma protein and b) lactate dehydrogenase ( LDH).

Statistics and calculations: Mean ± standard deviations are given in the Tables 1 and 2. For all three groups Initial-, Swim- and Rest-, the mean value of every measured parameter was compared pairwise. A Kruskal- Wallis test was performed on the data to check for significant differences between the three groups. Further statistics were performed using a one-way ANOVA. Comparisons of mean squares of the ANOVA were tested using F-tests. P≤ 0.05 was considered as statistically significant. Normality of the data and homogeneity of variances were checked by

Kolmogorov-Smirnov and Fmax tests, respectively.

PARAMETER Swim- Initial- Rest- P-value P-value P-value P-value group group group Kruskal Sw/In Sw/Re In/Re (N=9) (N=9) (N=12) -Wallis Body weight (g) 914.7± 688.8± 676.4± 0.0001 0.001 0.001 NS 58.37 113.4 70.94 Length (cm) 74.9± 67.2± 70.88± 0.009 0.002 0.008 NS 3.17 5.34 3.76 Hematocrit (%) 42.3± 32.1± 38.64± 0.0095 0.002 0.040 0.014 2.1 2.0 4.75 AAT (units/ml) 94.7± 106.1± 64.42± 0.001 NS 0.070 0.009 37.59 37.56 34.67 LDH (units/ml) 1300.1 507.4± 1381.2± 0.01 0.014 NS 0.032 ± 264.8 1073 818.3 Total protein (g/l) 42.0± 38.90± 43.83± 0.218 NS NS NS 12.95 6.80 3.21

Table 1: Test results (mean ±SD) among healthy (virus-negative) animals for the parameters body weight, length, hematocrit, aspartate aminotransferase (AAT), lactate dehydrogenase (LDH) and total protein. Comparison between groups: Swim-Initial (Sw/In), Swim-Rest (Sw/Re) and Initial-Rest (In/Re). NS indicates P-value ≥ 0.1

RESULTS

In this study, EVEX-virus, a rhabdovirus, was detected in Grevelingen animals which swam for a mean time period of 28.6 ± 7.7 days (range 14.0-40.0 days) and had covered a mean distance of 1,048 ± 278 km (range 526-1521 km).

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The mean distance covered was 36.9 ± 3.6 km per day (range 26.4-40.2 km). EVEX infected eel may show anemia, with blood in the abdominal fluid and hemorrhage all over the body. While the resting Initial group showed no signs of infection, the Swim-group developed severe hemorrhage and anemia at various time-points, which apparently forced them to stop swimming (Fig.1A).

PARAMETER Swim- Initial- Rest-group P-value P-value P-value P-value group group Kruskal Sw/In Sw/Re In/Re (N=14) (N=10) (N=13) Wallis Body weight (g) 1458.5± 1433.8± 1652.3± 0.564 NS NS NS 175.5 233.1 354.7 Length (cm) 85.4± 87.85± 89.44± 0.579 NS NS NS 8.7 3.94 7.34 Hematocrit (%) 7.03± 32.32± 34.01± 0.001 0.002 0.002 NS 4.3 7.34 7.99 AAT (units/ml) 2175± 98.83± 156.9± 0.0001 0.001 0.001 NS 2510 91.84 126.9 LDH (units/ml) 7290.9± 1771± 625.9± 0.0001 0.006 0.001 0.005 5447.9 921.3 775.0 Total protein (g/l) 24.19± 44.70± 41.3± 0.0001 0.001 0.002 NS 12.42 5.01 4.47

Table 2: Test results (mean ±SD) of infected (virus-positive) animals for the parameters body weight, length, hematocrit, aspartate aminotransferase (AAT), lactate dehydrogenase (LDH) and total protein. The units/ml for LDH and AAT correspond to µmol NADH/ml/min degraded to NAD+ at 37 ° C.Comparison between groups: Swim-Initial (Sw/In), Swim-Rest (Sw/Re) and Initial-Rest (In/Re). NS indicates P-value ≥ 0.1

All animals in Initial, Swim and Rest groups of the Grevelingen animals were infected with the EVEX-virus although the drop in hematocrit was only noticeable in the Swim group. The animals in the virus-negative-Swim group swam for a period of 173 days and covered a mean distance of 5,533 ± 342 km (range 4900-5949 km). The mean distance covered was 31.98 ± 2.05 km per day (range 28.3-34.4 km). In the group of non-infected hatchery animals the opposite effect was observed. The initial hematocrit value of 32.1 ± 2.0 % increased after 5500-km to 42.3 ± 2.1 % (Figure1B). To examine our hypothesis that a virus infection is the reason for the collapse, we tested plasma samples for LDH, total protein, and AAT (Tables 1 and 2).

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In Table 1 (healthy, virus-negative) and Table 2 (infected, virus-positive) the effect of swimming, the time aspect (endurance of the experiment with starving animals) and the influence of the virus was assessed. In the healthy group, swimming results had no effect on any of the selected parameters (Table 1). In the healthy group, a time effect results in a significant difference for AAT and LDH between Initial and Rest group. In the virus infected group this is only the case for LDH between Initial and Rest group. A clear virus effect can be observed in the infected Swim groups resulting in strongly significant different values of AAT, LDH and total protein in comparison with Initial and Rest group.

Discussion

In this study EVEX-virus was detected in a group of European eels used for long-term swimming experiments. While the Rest-group showed no signs of illness and looked healthy, the Swim-group showed signs of hemorrhage and severe anemia at different time-points, which apparently forced them to stop swimming (Fig. 1B). The hemorrhage took the form of petechial hemorrhages all over the body, and bloody abdominal fluid. Wolf (1988) described these defects as obvious signs of a severe viral infection. EVEX is the most prominent rhabdovirus, infecting eel-populations (Jørgensen et al. 1994, van Ginneken et al. 2004). Recently we found EVEX in European eel collected across its natural range: the Netherlands, Italy and Morocco. The virus was also observed in New Zealand longfin eel (A. dieffenbachi). In contrast, glass eel collected from eel farms in The Netherlands were mainly infected with HVA (Herpesvirus anguillae). Fish production via aquaculture has more than doubled in weight and value between 1986 and 1996 and over one quarter of human fish-consumption is produced in aquaculture (Naylor et al. 2000). With the growing amount of in aquaculture produced products, transfer of diseases by transport of stock and food supplies has increased. Blanc (1997) points out that nearly one hundred pathogens have been introduced in European hydrosystems since the introduction of aquaculture. Widespread infection of the eel-population with for instance EVEX virus may result from unlimited intercontinental transport. In a recent survey we found many viruses in eel populations in The Netherlands (van Ginneken et al. 2004), which threatens for the whole eel-population as The Netherlands is one of the leading eel-trading countries (Heinsbroek & Kamstra 1995).

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We therefore hypothesize that virus infections, probably easily spread due to increasing proximity with humans, may be a contributing factor to the worldwide decline of eel populations. We compared infected and non-infected animals for their spawning migration in large swim tunnels in the laboratory. Also we measured blood biochemistry data like hematocrit, total protein, AAT and LDH. We have to acknowledge that we used in this study eels of different life history stages. The infected eels were silver (migratory-stage), from a wild brackish water population, and with a size of around 1500 g. The non-infected eels were yellow (sedentary-stage) originating from a hatchery run on freshwater and with a size of around 700-900 g. The rationale behind this possible flaw in the experimental set-up was that the wild eel population seemed to be infected with the EVEX virus (van Ginneken et al. 2004). We were afraid to loose the eels in an early stage due to the EVEX virus, as for three consecutive years we tried to have a 5,500 km simulated migration run with Grevelingen eels in the swim tunnels. Every time the animals collapsed after 500-1,500 km. Eel farmers immunize their eels against eel viruses by means of bath exposure at the glass eel (elver) stage to adult, virus positive eels (personal communication with Ir.J.van Rijsingen, Royaal BV., Helmond, The Netherlands). We choose virus-resistant hatchery eels on freshwater for the 5,500 km run. To our opinion this difference between the two eel groups had no major impact on blood chemistry parameters. In the Rest-group there was (except for AAT) no significant difference between Grevelingen (infected) and hatchery (non-infected) animals giving P-values for total protein, AAT and LDH of respectively, P≤0.291, P≤0.019*, P≤0.052. In contrast in the Swim group the P-values between Grevelingen (infected) and hatchery (non-infected) animals for total protein, AAT and LDH were highly significant, respectively, P≤0.009**, P≤0.001**, P≤0.002**. This indicates that the initial status of the animals remained the same but that due to swimming exercise in combination with a virus infection there was a shift in blood chemistry parameters. Here, we have shown that LDH levels increased in the infected swim group. LDH is an abundant enzyme present in all tissues and is released upon tissue damage. Increased levels of LDH are associated with hemolysis, liver disease or hypoxemia due to a severe shock or anoxia (Burtis et al. 1996). Reduced total protein levels in the infected swim group suggest bleeding and/or liver failure. In principle, total plasma protein can be used as an indicator for disease for two reasons.

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First, most plasma proteins (with the exception of the immunoglobulins and protein hormones) are synthesized in the liver (Burtis et al. 1996). Therefore changed protein plasma levels may point to liver failure. Secondly, hemodilution can cause decrease of proteins. AAT is significantly higher in the infected Swim group. A small increase is normal after muscle exercise, but in this case the significantly increased level of AAT may suggest liver damage, hemolysis or general tissue damage. All three parameters: increased LDH, decreased total protein and increased AAT, are consistent with the hemorrhage seen in the animals. Possibly, the anemia is the result of internal blood loss, since the symptoms include blood in the abdominal fluid and hemorrhage all over the body. Acute human hepatocellular injury, whether due to viral hepatitis, hepatic ischemia or drug hepatotoxicity also resulted in elevated levels of serum AAT, while LDH was increased in case of ischemic hepatitis. It was concluded that these parameters may be helpful in the differential diagnosis of acute liver injury (Cassidy & Reynolds 1994). The second long-term swim trial of 5,500-km with the healthy virus-negative group, showed the opposite effect: the initial hematocrit value of 32.1% (±2.0) increased after 5500- km to 42.3% (±2.1) (Fig. 1B). Normal hematocrit values for healthy yellow vs. silver eel are 26.5 ± 1.0 % and 36.4 ± 1.2 %, respectively (Johansson et al. 1974).

For humans, long endurance exercise, resulting in a training effect with increased hematopoiesis, can probably be associated with the function of the hormone erythropoietin (Leigh-Smith 2004). The expression of an erythropoietin-like gene has been described in fish (Shiels & Wickramasinghe 1995). So, it is likely that, the increased hematocrit in the group that swam 5,500-km, could be mediated by erythropoietin. Infection with EVEX, and anaemia in healthy-looking adults, may have serious consequences for the population. As a catadromic fish species, eels have a very complex life cycle. The return journey of the adults of 5,500-km to their spawning grounds may be considered a stressful situation. If the changes observed in blood profile for European eel during a migration of 5,500-km in large swimtunnels in the laboratory also happen in EVEX infected eels in the ocean, this may have serious consequences for the adult silver eel during their natural migration.

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Acknowledgements We thank Prof. M. Richardson (Leiden University, The Netherlands) for helpful suggestions and improving the manuscript. This work was supported by the Foundation for Technical Research (LBI.4199), which is subsidized by the Netherlands Organization for Scientific Research (N.W.O.) and the European Commission, project (EELREP, Q5RS-2001-01836).

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Haenen, O.L.M.; Banning van, P.; Dekker, W., 1994. Infection of eel Anguilla anguilla (L.) and smelt Osmerus eperlanus (L.) with Anguillicola crassus (Nematoda, Dracunculoidea) in the Netherlands from 1986 to 1992. Aquaculture 126, 219-229. Heinsbroek, L.T.N. and Kamstra, A.,1995. The River Eels, Chapter 6. In: Nash, C.E., Novotny, A.J. (Eds.), Production of Aquatic Animals, Elsevier, Amsterdam pp. 109-131. Johansson, M.L.; Dave, G.; Larsson, A.; Lewander, K.; Lidman, U., 1974. Metabolic and Hematological studies on the yellow and silver phases of the European eel, Anguilla anguilla L. III. Hematology. Comp.Biochem.Physiol. B 47, 593-599. Jørgensen, P., Castric, J., Hill, B., Ljungberg, O. and De Kinkelin, P., 1994. The occurrence of virus-infections in elvers and eels (Anguilla anguilla) in Europe with particular reference to VHSV and IHNV. Aquaculture 123, 11-19. Klauke, R.; Schmidt, E.; Lorentz, K., 1993. Recommendations for carrying out standard ECCLS procedures (1988) for the catalytic concentrations of creatine kinase, aspartate aminotransferase, alanine transferase and gamma-glutamyltransferase at 37 °C. Eur.J.Clin.Chem.Clin.Biochem. 31, 901-909. Knights, B., 2003. A review of the possible impacts of long-term oceanic and climate changes and fishing mortality on recruitment of anguillid eels of the Northern Hemisphere. The Science of the Total Environment 310, 237-244. Kobayashi, T. and Miyazaki, T., 1996. Rhabdoviral dermatitis in Japanese eel, Anguilla japonica. Fish Pathol. 31, 183-190. Leigh-Smith, S., 2004. Blood boosting. Brit.J.Sport.Med. 38, 99-101. Miller, M.; McCleave, J.D., 1994. Species assemblages of leptocephali in the Subtropical Convergence Zone of the Sargasso Sea. Journal of Marine Research 52, 743-772. Naylor, R.L.; Goldburg, R.J.; Primavera, J.H.; Kautsky, N.; Beveridge, M.C.C.; Clay, J.; Folke, C.; Lubchenco, J.; Mooney, H.; Troell, M., 2000. Effect of aquaculture on world fish supplies. Nature 405, 1017-1024. Sano, T., Nishimura, T., Okamoto, N. and Fukuda, H., 1977. Studies on viral diseases of Japanese fishes. VII. A rhabdovirus isolated from European eel (Anguilla anguilla). Fish Pathology 10, 221-226. Schmidt, J. , 1923. Breeding places and migration of the eel. Nature 111, 51-54. Shiels, A.; Wickramasinghe, S.N., 1995. Expression of an erythropoietin-like gene in the trout. Brit.J.Haematol. 90, 219-221.

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Stone, R., 2003. Freshwater eels are slip-sliding away. Science 302, 221-222. Svedäng, H.; Wickström, H., 1997. Low fat contents in female silver eels: indications of insufficient energetic stores for migration and gonadal development. J.Fish Biol. 50, 475-486 . Tesch, F.W., 1977. "The eel", Biology and management of anguilled eels, Chapman & Hall, London 434 pag. ISBN 0-412-14370-4. Ueno, Y., Kitao, T., Chen, S-N., Aoki, T. and Kou, G-H., 1992. Characterization of a Herpes-like Virus isolated from cultured Japanese eels in Taiwan. Gyobyo Kenkyu 27, 7-17. van den Thillart , G.; van Ginneken V.; Körner F.; Heijmans, R.; van der Linden, R. and Gluvers, A., 2004. Endurance swimming of European eel. J.Fish Biol. 65, 1-7. van Ginneken, V.; Haenen, O.; Coldenhoff, K.; Willemze, R.; Antonissen, E.; van Tulden, P.; Dijkstra, S.; Wagenaar, F.; van den Thillart, G., 2004. Presence of Virus infections in Eel populations from various geographic areas. Bulletin European Association of Fish Pathologist , 24(5), 270-274. van Nieuwstadt, A.P., Dijkstra, S.G. and Haenen, O.L.M., 2001. Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla. Dis. Aquat. Org. 45, 103-107. Wolf, 1988. K. Fish viruses and fish viral diseases. Cornell University Press, Ithaca, USA, Comstock Publishing Associates, New York, 476 pp; 1988.

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Presence of eel viruses in eel species from various geographic regions.

V.van Ginneken1*, O Haenen2, K.Coldenhoff1, R.Willemze3, E.Antonissen1, P.van Tulden2, S.Dijkstra2, F.Wagenaar2 and G. van den Thillart2

1: Institute Biology Leiden, Leiden University, The Netherlands 2: Central Institute for Animal Disease Control (CIDC-Lelystad), Lelystad, The Netherlands 3: Leiden University Medical Center, Hematology, Leiden University, The Netherlands

To whom correspondence should be addressed. E-mail: [email protected]

Published in: Bulletin European Association of Fish Pathologist (2004) 24(5): 270-274.

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De generatio spontanea: paling onstaat uit de huid van paling, de “aels-vellen” Laten wij stellen, dat van twintig of meer Alen, die men het vel sal af halen, een vande selvige is, welkers jongen inde Baar-moeder, al soo verre gekomen, sijn, dat die uijt de Baar-moeder gestooten werden. Wanneer men nu soo danigen Ael het vel aftrekt oft afstroopt, in welk doen men het vel dubbelt over het lighaam haalt, soo werd in dat doen, het lighaam vanden Ale, meer als gemeen gedrukt, door welke drukkinge, veel jonge Alen, uijt de Baarmoeders konnen gestooten werden, en nog meer als de huijt vande Alen aan de opening vande excrementen, en Baar-moeder, komt ontstukken te schueren, als wanneer men dan met de hand, of ook wel met het mes, een stuk vanden uijtgang, komt af te snijden, en dus nog meerder de Baar- moeder komt te parssen. Dese uijt gedrukte jonge Alen aan de vellen blijven hangen, ofte van binnen in het vel, dat met het afhalen om gekeert is, sijnde, en soo verre gekomen wesende, dat die uijt het water haar voetsel konnen trekken, indien dan dese vellen int water geworpen werden, in een nieuw gedolven sloot, soo konnen daar Alen van voort komen. . (Antoni van Leeuwenhoek, Brief No. 123 [75], 16 september 1692).

Chapter 11

Presence of eel viruses in eel species from various geographic regions

EVEX (Eel-Virus-European-X), HVA (Herpesvirus anguillae), and EVE (Eel Virus European) were detected in wild and farmed European eels (Anguilla anguilla L.) from the Netherlands, EVEX and EVE from farmed eels from Italy, and EVEX from wild eels from Morocco. EVEX was also isolated from wild New Zealand eel (A. dieffenbachi). Elvers (A.anguilla) collected from eel farms in the Netherlands were mainly infected with HVA. An unknown picornavirus was isolated from healthy wild European eel from The Netherlands, and New Zealand eel (A.dieffenbachi) from New Zealand. European eels from Northern Ireland showed clinical signs of virus infections although no viruses were detected in these samples.

Fish viruses can cause disease or even mortality when fish are under stressful conditions. Eels can suffer various viral infections. Firstly, infections with EVA (Eel-Virus-America) and EVEX have been described. Both viruses are serologically related (Kobayashi & Miyazaki 1996). EVA was first discovered in Japan in 1974, in a shipment of American elvers, which had been stocked in Cuba (Wolf, 1988). Another virus, which was isolated in a shipment from France to Tokyo, was named EVEX because of its European origin (Sano et al., 1977). Another eel virus is EVE (Eel Virus European). Egusa (1970) described a new disease of Japanese eels (Anguilla japonica), called branchionephritis, with mortalities up to 50%. The eels had had contact with newly imported infected European eels (A. anguilla). In 1976 the EVE (European Virus of Eel) was isolated for the first time (Sano, 1976) from European eels, showing severe renal pathology. The virus appeared to be serologically close by related to the French isolate of Infectious Pancreatic Necrosis Virus (IPNV), d'Honnincthun (Sano et al., 1981). Okamoto et al. (1983) regrouped EVE to group III of IPNV, together with the Danish isolate of IPNV, strain Ab (Jørgensen, 1971). The French isolate was placed in group II of IPNV, by the same authors. Hedrick et al. (1983) stated, that both EVE and IPNV type Ab were close related, but unique virus strains. EVE was pathogenic for Japanese eels, but not for rainbow trout fry (Sano et al, 1981). Another eel virus is HVA Herpesvirus anguillae . In Asia, (HVA) it was isolated in 1985 from diseased Japanese eel Anguilla japonica and European eel A. anguilla (Sano et al. 1990), showing skin and gill erythema and necrosis of skin, gills, and liver.

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HVA is known to occur also in farmed European eel A.anguilla, where it has caused considerable disease problems (Davidse et al., 1999; Haenen et al., 2002). The aim of this study was to investigate the presence of viruses in eel populations from various geographic regions.

Live eels (25 lots) originating from 9 different countries (Table 1) were collected via an eel importer at Schiphol airport (The Netherlands). On the day of arrival they were anaesthetized with 300 ppm MS222 (3-aminobenzoic-acid-ethyl-ester methane sulphonate salt, Sigma, St. Louis, USA). Spleen, gills, kidney and liver were sampled and stored on dry ice. For virus isolation, at the Fish Diseases Laboratory (CIDC-Lelystad), samples of organs were homogenized with sterile medium and sterile sand, and inoculated on three cell-lines: RTG-2 - Rainbow Trout Gonad cells, FHM - Fat Head Minnow cells, and EK-1 - Eel Kidney cells, at 15°C, 20°C, and 26°C respectively, according to standard procedures (Davidse et al., 1999). In case of cytopathic effect, the infected cell line was inspected by electron microscopy followed by immunofluorescence or immunoperoxidase methods in order to identify the virus (Wolf, 1988, Davidse et al., 1999).

Several viruses were isolated from the investigated samples. Predominantly, EVEX was detected in wild and farmed European eel from various geographic regions: The Netherlands, Italy, and Morocco. However, only the eels from Italy showed clinical signs, like haemorrhages and red skin areas. Apart from European eel, EVEX was also isolated from wild New Zealand eel from New Zealand, although they had no clinical signs of disease. HVA was detected in farmed glass eels (elvers of A.anguilla) in The Netherlands, and in farmed adult eels in Italy from pond culture. In all these cases, the eels showed clinical signs of disease, like reddening with petechial haemorrhages. HVA is known to be present in farmed Japanese eels (A.japonica) in Taiwan (Ueno et al., 1992, 1996; Chang et al., 2002) and in farmed European eels in The Netherlands (Davidse et al., 1999, Haenen et al., 2002). In this study, we isolated HVA for the first time from wild (adult) European eels, in The Netherlands. It supports the hypothesis of Van Nieuwstadt et al (2001) and Haenen et al. (2002), that HVA is wide spread.

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Country Location Species Clinical signs Virus positive at Infection rate incubation temp (°C) (no. of eels) NETHERLANDS Lake Grevelingen A.anguilla - EVEX 15 and 20 6 from 6 Lake Grevelingen A.anguilla + HVA 20 and 26 1 from 10 Lake Grevelingen A.anguilla - EVEX 15 and 20 1 from 2 Lake Grevelingen A.anguilla - picorna-like 20 1 from 2 Lake Brasemer A.anguilla - negative - 0 from 10 Lake Brasemer A.anguilla - negative - 0 from 10 Lake Lauwers A.anguilla - negative - 0 from 10 Lake Lauwers A.anguilla + HVA 20 and 26 10 from 10 Eel farm-1 A.anguilla + HVA 20 and 26 pooled elvers Eel farm-2 A.anguilla + HVA 20 and 26 pooled elvers Eel farm-3 A.anguilla + HVA 20 and 26 pooled elvers ITALY Eel farm-4 A.anguilla - EVEX & EVE 15, 20 and 26 4 from 4 Eel farm-5 A.anguilla + EVEX 20 and 26 3 from 4 FRANCE Loire A.anguilla - negative - 0 from 4 Perpignan A.anguilla + negative - 0 from 3 MOROCCO Sebou A.anguilla - EVEX 26 2 from 4 USA Virginia A.rostrata - negative - 0 from 4 CANADA St.Lawrence A.rostrata - negative - 0 from 4 NEW ZEALAND Tekawata A.dieffenbachi - picorna-like 20 1 from 4 Tekawata A.dieffenbachi - EVEX 26 1 from 4 IRELAND River Roosky A.anguilla + negative - 0 from 4 Lake Corrib A.anguilla + negative - 0 from 4 Lake Ennel A.anguilla + negative - 0 from 4 mixed origin A.anguilla + negative - 0 from 4 UN. KINGDOM Lake Erne (North. Ireland) A.anguilla + negative - 0 from 4

Table 1. Eel viruses isolated from eels of different locations. EVEX: Eel-Virus European X, HVA: Herpesvirus anguillae, EVE: European Virus of Eel

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The impact of the isolated picorna-like virus from both European eels from The Netherlands and New Zealand eel (A.dieffenbachi) from New Zealand is not known, as they did not show clinical signs of disease. The adult European eels from Northern Ireland showed clinical signs of disease, like petechial haemorrhages, and red skin areas, but no virus was isolated from these eels. We isolated several viruses from three eel species from various regions. Even elvers, for restocking were found positive for virus (HVA). Jørgensen et al. (1994) studied elvers and eels (A.anguilla) from several European countries (Denmark, England, France and Sweden) for presence of virus. They isolated EVEX, EVA, IPNV (Infectious Pancreatic Necrosis virus), and herpes-like viruses. There is much trade in live eels on a global scale. Trade of eels from infected areas like The Netherlands, which is one of the leading eel trading countries (Heinsbroek & Kamstra, 1995), could impose a risk to other eel populations by transmitting several eel viruses.

ACKNOWLEDGMENTS

This study was supported by the Netherlands Organisation for Scientific Research (STW-project no. LBI66.4199) and by the European Commission (project QLRT-2000-01836). Professor R. Willemze is acknowledged for providing funds for performing the virus detections.

REFERENCES • Chang, P.H., Pan, Y.H., Wu, C.M., Kuo, S.T. and Chung, H.Y., 2002. Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan. Dis. Aquat. Org. 50,111-118. • Davidse, A., Haenen, O.L.M., Dijkstra, S.G., van Nieuwstadt, A.P., van der Vorst, T.J.K., Wagenaar, F. and Wellenberg, G.J.,1999. First Isolation of Herpesvirus of Eel (Herpesvirus Anguillae) in diseased European eel (Anguilla anguilla L.) in Europe. Bull.Eur.Ass.Fish.Pathol. 19, 137-141. • Egusa, S., 1970. Branchionephritis prevailed among eel populations in farm-pond in the warm water 1969-1970. Fish Pathol. 5 , 51-56 (In Japanese). • Haenen, O.L.M., Dijkstra, S.G., van Tulden, P.W. , Davidse, A., van Nieuwstadt, A.P., Wagenaar, F. and Wellenberg, G.J., 2002. Herpesvirus anguillae (HVA) isolations from

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disease outbreaks in cultured European eel, Anguilla anguilla in The Netherlands since 1996. Bull.Eur.Ass.Fish Pathol., 22(4), 247-257. • Hedrick, R.P., Fryer, J.L., Chen, S.N., and Kou, G.H. ,1983. Characteristics of four birnaviruses isolated from fish in Taiwan. Fish Pathol. 18(2), 91-97. • Heinsbroek, L.T.N. and Kamstra, A.,1995. The River Eels, Chapter 6. In: Production of Aquatic Animals (C.E., Nash and A.J. Novotny, Eds.) pp. 109-131. Elsevier, Amsterdam. • Jørgensen, P.E.V. , 1971. Problems in the serological typing of IPN virus. Acta vet. Scand. 12, 145-147. • Jørgensen, P., Castric, J., Hill, B., Ljungberg, O. and De Kinkelin, P.,1994. The occurrence of virus-infections in elvers and eels (Anguilla anguilla) in Europe with particular reference to VHSV and IHNV. Aquaculture 123,11-19. • Kobayashi, T. and Miyazaki, T.,1996. Rhabdoviral dermatitis in Japanese eel, Anguilla japonica. Fish Pathol. 31, 183-190. • Okamoto, N., Sano, T., Hedrick, R.P., and Fryer, J.L. ,1983. Antigenic relationships of selected strains of infectious pancreatic necrosis virus and European eel virus. J. Fish Dis. 6, 19-25. • Sano, T. ,1976. Viral diseases of cultured fishes in Japan. Fish Pathol. 10, 221-226. • Sano, T., Nishimura, T., Okamoto, N. and Fukuda, H.,1977. Studies on viral diseases of Japanese fishes. VII. A rhabdovirus isolated from European eel (Anguilla anguilla). Fish Pathol. 10, 221-226. • Sano, T., Okamoto, N., and Nishimura, T., 1981. A new viral epizootic of Anguilla japonica Temminck and Schlegel. J. Fish Dis. 4, 127-139. • Sano, M., Fukuda, H. and Sano, T., 1990. Isolation and characterization of a new herpesvirus from eel. In: Pathology in Marine Science (Perkins, T.O. and Cheng T.C., Eds.), 15-31. Academic Press, New York. • Schmidt, J.,1923. Breeding places and migration of the eel. Nature, 111, 51-54. • Van Nieuwstadt, A.P., Dijkstra, S.G. and Haenen, O.L.M., 2001. Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla. Dis. Aquat. Org. 45, 103-107. • Ueno, Y., Kitao, T., Chen, S-N., Aoki, T. and Kou, G-H.,1992. Characterization of a Herpes-like Virus isolated from cultured Japanese eels in Taiwan. Gyobyo Kenkyu 27, 7- 17.

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• Ueno, Y., Shi, J.-W., Yoshida, T., Kitao, T., Sakai, M., Chen, S.-N. and Kou, G.H.,1996. Biological and serological comparisons of eel herpesvirus in Formosa (EHVF) and herpevirus anguillae (HVA). J.Appl.Ichthyol. 12, 49-51. • Wolf, K.,1988. Fish viruses and fish viral diseases. Cornell University Press, Ithaca, USA, Comstock Publ. Assoc., New York, 476 pp.

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Effects of PCBs on the energy cost of migration and blood parameters of European silver eel (Anguilla anguilla, Linnaeus 1758)

Vincent van Ginneken1, Arjan Palstra1, Pim Leonards3, Maaike Nieveen1, Hans van den Berg2, Gert Flik4, Tom Spanings4, Patrick Niemantsverdriet1, Tinka Murk2, Guido van den Thillart1,

1) Department of Integrative Zoology, Institute of Biology Leiden, van der Klaauw Laboratories, P.O.Box 9511, 2300 RA Leiden, The Netherlands. 2) Toxicology section, Wageningen University, Tuinlaan 5,PO Box 8000, 6700 EA, The Netherlands. 3) Netherlands Institute for Fisheries Research (RIVO), P.O. Box 68, 1970 AB IJmuiden, The Netherlands 4) Department of Animal Physiology, Institute for Neuroscience, Faculty of Science, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands

Corresponding Author: Dr.Guido van den Thillart, Department of Integrative Zoology, Institute of Biology Leiden, van der Klaauw Laboratories, P.O.Box 9511, 2300 RA Leiden, The Netherlands. E-mail: [email protected]

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bestudering van de bloedsomloop in een paling Ik kan niet na laten bij dese gelegentheijt, tot UE. Hoog Edele te seggen, dat ik voorleden jaar heb levend gehouden, wel drie maanden lang, kleijne Aeltgens, waar van de grooste niet langer waren als een vinger. Dese Aeltgens waren in een groote platbodemde, Aerde, verglaasde pot, en geplaast in mijn kelder, die seer koel is, en ten minsten eens ter week met vers water besorgt. Wanneer nu inde Na Sommer, eenige Heeren mij quamen besoeken, en versorgten, de Circulatie van het Bloet te sien, liet ik eenige vande geseijde Aeltgens tot mij brengen, deselve beschouwende, konde ik int eerst geen loop van het Bloet gewaar werden, dat mij vreemt voor quam, te meer, om dat de Aeltgens soo vlugge int voort swemmen waren, als of deselve eerst gevangen werden. Eijntelijk, wierde ik soo nu en dan, een enkel bolletge Bloet inde dunste Bloet-vaaten gewaar, en dus konde ik de Heeren de Bloed niet laten sien. Bij mijn selven sijnde, beschouwde ik verscheijde malen de verhaalde Aeltgens, met deselve uijtkomst. Dog wanneer ik mijn oog liet gaan, op de grooste Bloet-vaaten die voor mij te ontdekken waren, sag ik dat de loop van het Bloet, met de gewoonelijke snelte was loopende, om dat de bolletgens inde groote vaaten, mij in meerder getal voor de oogen quamen, dog op het honderste deel soo veel niet, dan of de Aeltgens eerst gevangen waren, waar uijt ik in gedagten nam, dat de Bloet bolletgens, door gebrek van spijs, meest alle tot voedsel van het lichaam waren overgegaan, en ik liet de Aeltgens int water werpen, als onnut voor mij om dienst te doen, en om dat ze voetsel soude konnen bekommen. (Antoni van Leeuwenhoek, Brief No. 228[140], 2 augustus 1701). Chapter 12

Effects of PCBs on the energy cost of migration and blood parameters of European silver eel (Anguilla anguilla, Linnaeus 1758)

Abstract

The effect of polychlorinated biphenyls (PCBs) on the energy consumption of fasting silver European eel (Anguilla anguilla L.) was studied over a 27 days period during which the animals were at rest or were swimming 800 km in Blazka swim tunnels. The fish were dosed intraperitoneally with a PCB-mixture consisting of 5 mg/kg eel PCB-153, 7 μg/kg eel PCB- 126 and 50 μg/kg eel PCB-77 or only with the vehicle corn oil (10 ml/kg, controls). The results revealed five major observations: First, PCB-exposed animals loose less weight and have lower glucose and cortisol (only swimming) levels compared to their unexposed controls. Second, PCB-concentrations on a lipid basis are 2.7 times higher in swimming compared to resting animals. Third, PCB-exposure significantly reduces oxygen consumption during swimming of the PCB-exposed animals from 400 km on (18 days) and this effect -1 -1 increases with time. The Cost of Transport (COT, [mg O2. kg . km ]) is significantly lower in PCB exposed animals from 100 km up to 800 km. In addition the standard metabolic rate measured 2 days after the last swimming activity is significantly lower in the PCB-exposed animals. Fourth, the spleen is increased in the PCB-exposed swim animals but not in the PCB-exposed Control animals. Fifth, silver eels easily survive resting in marine water and forced swimming in fresh water, but not in a combination of these two stress factors. Plasma- pH, ion levels (sodium and potassium), plasma lactate acid, haemoglobin and hematocrit were unaffected by PCB-exposure. We conclude, that PCB-exposure interferes with the energy metabolism of silver eel in marine water and appears to interfere with cortisol control over (carbohydrate) metabolism. This effect was greater in swimming than in resting eel.

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Introduction

Since the late 70's of the last century the recruitment of the European eel population (Anguilla anguilla L.) has been dwindling. A dramatic decrease has been observed in the yearly number of glass eel entering European freshwater (Dekker 2004). Also steep declines of biomass of glass eel of 90-99% have been reported for other eel species like the Japanese eel (Anguilla japonica), and American eel (Anguilla rostrata) (Castonguay et al. 1994, Stone 2003). Eel is a catadromic fish species with their spawning areas thousands of kilometres away in the ocean. As a result sufficient energy reserves and an efficient metabolism is critical for this species. Possible causes for the decline of eel populations include oceanographic changes (Knights 2003), overfishing (Dekker, 2005), viruses (van Ginneken et al. 2004, 2005b) or swim bladder parasite (Haenen et al. 1994). Eels may be very vulnerable to persistent toxic hydrophobic contaminants that are released during migration and which may interfere with energy metabolism and reproduction. In eels, originating from inland waters, high levels of especially polychlorinated biphenyls (PCBs) have been reported repeatedly in ranges 1.5-10 mg/g (De Boer & Hagel 1994), therefore regularly exceeding the Dutch standards for human consumption which is 0.5 mg/kg for PCB 153 for eel. Polyhalogenated aromatic hydrocarbon (PHAH) pollutants, including PCBs and polychlorinated dibenzo dioxins/-furans (PCDD/Fs, further referred to as ‘dioxins’), are lipophilic, persistent and widely spread in the environment and known to accumulate in the food chain. It is estimated that over 30% of the one million tons of PCBs produced are still present in aquatic and terrestrial ecosystems (Voltura and French 2000). Exposure to PHAHs, especially ‘dioxins’ and dioxin-like PCBs has been shown to lead to adverse effects in many species, including disturbance of retinoid and thyroid hormone homeostasis, adverse effects on male and female reproduction, developmental toxicity, hepatotoxicity, immunotoxicity, progressive weight loss (‘wasting syndrome’) and tumor promotion (Goldstein and Safe, 1989; Mukerjee, 1998; DeVito and Birnbaum, 1994). Little is known about effects of PCB exposure in combination with activity and especially on spawning migration of eels. During this period the starving eel relies completely on its energy stores in the adipose tissues. PCBs stored in fat will be mobilised when fat is used, increasing the internal concentrations. It is therefore hypothesised, that the release of PCBs from fat stores may interfere with eel physiology and energy metabolism thus impairing the capacity to reach the spawning grounds.

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In this study we exposed eel to PCBs 10x the amount accepted for fish consumption. The PCB-mixture consisted of a di-ortho-, planar- and metabolisable PCB in a relative and absolute amount which is environmentally relevant. Effects were studied on their energy metabolism, PCB-levels and blood parameters during a simulated migration over 800 km in Blazka swim tunnels compared to the effects on similarly exposed resting eel.

Material & methods

Animal treatment and husbandry conditions Three-year-old female hatchery eels (silver stage) between 73-80 cm long weighing around 1 kilogram were obtained from an eel farm in The Netherlands (Royaal BV, Helmond, The Netherlands). Eels were transported to the Netherlands Institute for Fisheries Research (RIVO, IJmuiden, The Netherlands), where they were gradually adapted from fresh to natural sea water over a period of four weeks. After this adaptation period of four weeks 44 eels were IP injected with the PCB-mixture in corn oil or with corn oil only (controls, 10 ml/kg). Thereafter eels were kept there for two weeks in a recirculation system with sea water to allow the animals to adapt to their new environment and the PCBs to distribute over the body. Thereafter the animals were transported to Leiden University. Upon arrival in Leiden they were anaesthetised with 100 mg/l benzocaine, weighed, measured (length, eye index), and tagged with a Trovan ID-100 implantable transponder microchip in a biocompatible glass capsule in the dorsal muscle 10 cm behind the head of every eel. To prevent skin infections the eels were treated with the antibiotic Flumequine (50 mg/L) for 3 h in a separate tank before transfer to the experimental units. Immediately hereafter the animals were individually assigned to either one of the 22 swim-tunnels of 127 L or twenty-two 50L flow-through containers. The eels were stimulated to swim for 750-800 km in the swim-tunnels in approximately 27 days, or left rested individually in the 50L flow-through boxes for the same period. Tunnels and containers all were connected to the same water recirculation system of

6,000 L of artificial sea water (Instant Ocean, Smulders B.V., Maastricht). The NH3 and NO

value of the water was checked daily. At values above 0.1-ppm NH3 and/or 0.1-ppm NO the water was refreshed from a separate 3,000-L storage tank. Temperature of the water was 19.0° ± 0.3 ° C. Experiments were performed in infra red light 670 nm light (bandwidth 20 nm) which is invisible for eel (Pankhurst & Lythgoe 1983).

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Blazka swim-tunnel: The Blazka swim-tunnel has a length of 200 cm, a diameter of the outer swim-tunnel tube of 28.8 cm and a diameter of the inner swim-tunnel tube of 19.0 cm. The volume was 127.14 ± 0.90 liter (n=5). It was calibrated with a Laser Doppler technique at the Delft Hydraulics Laboratory, Technical University Delft. The experimental set-up is described elsewhere (van den Thillart et al. 2004). The water flow in the swim tunnels was set for each animal at 0.5 body length per second ( ca 0.4 m/s).

Oxygen consumption measurement With an oxygen electrode (Mettler, Toledo), the oxygen level in each tunnel was measured continuously. The oxygen consumption rate was calculated from the oxygen decline after automatic closure of the water-inlet by a magnetic valve. The oxygen levels varied between 85 and 75% air saturation. The valve was normally open allowing a refreshment rate of 5-7 l.min-1 and automatically closed between 14.00 and 17.00 hours to measure oxygen consumption. The oxygen value was not allowed to fall below 75%, at that moment the valve automatically opened again, in order to avoid hypoxia (van den Thillart and van Waarde, 1985). From the

decrease in O2-concentration, the rate of oxygen consumption (VO2) was calculated following -1 -1 -1 -1 the formula: VO2 = 127.Δ[O2].Δt (mgO2.h .kg ), where: Δ[O2].Δt is the decrease of the oxygen content per hour (van den Thillart et al. 2004). Oxygen consumption data were corrected for body mass assuming a linear decline in mass of the animals between start and end of the experiment.

Exposure protocol The resting and the swimming group were each divided into two exposure groups: a control and a PCB-exposed group, in total 4 groups of 11 animals each. The eels in PCB group were injected intraperitoneally (IP) with a mixture of PCB-153 (5 mg/kg ww), PCB-126 (7 μg/kg ww), and PCB-77 (50 μg/kg ww) dissolved in corn oil. These levels were calculated as 10x the PCB-standard for food consumption in 2001 of 0.5 mg (sum of 7) PCBs/kg eel given as the most common PCB-153; 70 ng TCDD-equivalent (TEQ)/kg eel, dosed as the most relevant planar PCB126 (with a TCDD equivalency factor (TEF) of 0.1) 700 ng (0.7 ug) PCB126/kg; plus PCB-77 as a representative of metabolisable PCBs in an average environmental ratio to PCB-126: 5 ug PCB-77/kg eel.

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The internal dose of the animals is determined after termination of the experiment in filet and plasma samples using a reporter gene assay for bio-analysis of dioxin-like toxic potency (Murk et al., 1998).

Standard Metabolic rate and termination of the experiment After swimming 800-km the Swim-Control and Swim-PCB group were allowed to rest for 24 h and resting oxygen consumption was thereafter measured for a period of 4 hours as an estimate of the Standard Metabolic Rate (SMR) (Fry 1971). Hereafter the animals were anaesthetised and blood was collected with a heparinized syringe. After the animals were sacrificed several external parameters and blood parameters were determined and tissues collected. After the swim experiment the Rest-Control and Rest-PCB group were placed in the tunnels, after a 2 day habituation the resting oxygen consumption was measured for a period of 4 hours. Thereafter the eels were sacrificed and analysed as for the swim group.

Fat extraction from muscle samples for determination of the internal dose Muscle samples for bio-analysis of TEQ-values were homogenized by scraping and mincing with a sharp razor. To a portion of 1 gram of this thick muscle homogenate 2 ml water was added and for 1 minute further homogenized with a high speed mixer (Ultra Thurrax T25, Janke & Kunkel, Germany) after which 2 ml of isopropyl alcohol was added. This mixture was sonicated for 10 minutes and a liquid/liquid extraction was performed with 3 ml of hexane /di-ethylether (97:3, v/v). The extraction was repeated twice after the addition of 2 drops of concentrated hydrochloric acid to enhance the extraction. The organic layers were

pooled and evaporated under a gentle stream of N2 at 30°C. The amount of fat extracted was determined gravimetrically. For the clean up of the extracts a multi-layer sulphuric acid silica

column was used consisting of 1 g Na2SO4 on top of 2 grams of dried silica with 10% hexane pre-washed sulferic acid, 4 grams of dried silica with 20 % H2SO4 and 4 grams of dried

silica with 33% H2SO4. The column was eluted with 40 ml of hexane: diethyl ether (97:3,

v/v) as described for sediment samples by Murk et al., (1998). The eluate was dried with N2 at 30°C and 12 μl of DMSO was added just before complete evaporation of the organic solvent.

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Bio-analysis of TEQs in a reporter gene assay The DR-CALUX (Dioxin Receptor-mediated Chemical Activated LUciferase eXpression) assay was performed using H4IIE.Luc-cells in white 96-well view plates (Packard) according to the method developed by Murk et al. (1998). This assay is based on activation of the Arylhydrocarbon (Ah)-receptor by dioxin-like chemicals, which results in transcription and translation of the newly introduced gene for luciferase activity. Of the 3 PCB-congeners dosed in this experiment de DR-CALUX quantifies PCB-126 (which is not metabolized by the eel) and the small amount of PCB-77 that could be left after 6 weeks. PCB-153 is not active in the DR-CALUX. Luciferase activity is quantified in a luminometer after addition of the substrate luciferine. Cells were exposed in triplicate to eel muscle extracts in DMSO (0.4% max). For the quantification of induced responses on each microtiter plate, a concentration series of the reference compound 2,3,7,8-TCDD was included and 1-site-ligand curve fitted was performed using Slidewrite 6.0. The limit of quantification was set at the DMSO-response plus 3 times the standard deviation. After correction for the background signal of the DMSO solvent control, luciferase activities of sample dilutions interpolated on this curve and expressed as TCDD Equivalents (TEQ)-values per gram lipid when the response was between 0.9 and 6 pM TCDD.

Analysis of blood parameters Blood was collected by puncture of the caudal vessels with a heparinized (Leo Pharmaceuticals products, Ltd) tuberculin syringe fitted with a 25 Gauge needle. Haemoglobulin content in 20 μl blood was detected after 3 minutes using the cyan-methaemo- globulin method (Boehringer Mannheim, F.R.G.). Plasma was obtained by centrifugation in an Epperdorf centrifuge for 5 min at 13.000 g and divided over several cups as required for further analyses. Hematocrit was measured directly in 9 μl whole blood sample using a hemato- crit micro-centrifuge (Bayer, FRG). Plasma potassium was determined by flame photometry (Radiometer Copenhagen FLM3). Plasma was further analyzed with a Stat Profile pHOx Plus analyser with automated two-point calibration; this analyzer provides electrode readings for Na+, Ca2+, pH, and is equipped with enzymatic electrodes (Nova Biomedical, Waltham MA, USA) for glucose and lactate. Plasma cortisol was determined by radioimmunoassay as described by Arends et al. (1998).

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Statistics and calculations Mean values ± standard deviation (SD) are presented of 6-7 animals unless otherwise indicated. The eye-index (E.I.) was calculated according to the method of Pankhurst (1982): E.I.={[A+B)2/4*π]/L}* 100; where A is the horizontal eye diameter, B is the vertical diameter, and L is the total body length (mm). The Hepato somatic Index (H.S.I.) was calculated according to {[Liver weight]/[Body weight]}* 100%. The Spleen somatic Index (S.S.I.) was calculated according to {[Spleen weight]/[Body weight]}* 100%. The Gonado somatic Index (G.S.I.) was calculated according to {[Ovary weight]/[Body weight]}* 100%. Statistical analysis of the results was performed by SPSS software. Data were subjected to a one way ANOVA and mean squares of the ANOVA were compared using F-tests. P≤ 0.05 was considered as statistically significant. Normality distribution of the data was assessed and homogeneity of variances were checked by Kolmogorov-Smirnov and Fmax tests, respectively. Because the cortisol data were not normally distributed due to large variance these data were tested following a Mann-Whitney procedure. In order to test if the different calculated index parameters (H.S.I., G.S.I. and S.S.I.) were influenced by the somatic weight of the fish, a covariance analysis (ANCOVA) was performed in SPSS using a General Linear Model. These indices were also not normally distributed and consequently tested using a Mann-Whitney procedure.

Results

General condition and performance At the start of the experiment the female animals were at the silver stage, weighing between 900 and 1100 g, and with E.I. ranging 10.0-10.7. The recorded swimming distances at 27 days, when the experiment was terminated, were not significantly different with 767 ± 112 km for the PCB-treated eels (n = 7) and 781 ± 124 km for the controls (n = 6; P> 0.10) (table 1). The weight loss during this period was 70% and 72% for the PCB-exposed animals compared to Control animals in respectively the swimming and the resting groups (swim-rest PCB P ≤ 0.09, swim-rest Control P ≤ 0.12 (table 1, figure 1a)). The weight loss in the swim groups (PCB vs. Control) was not significantly different (P ≤ 0.34). Also for the rest groups (PCB vs. Control) there was no significant difference in weight loss (P ≤ 0.24).

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Organs and tissue: PCB-levels in muscle from the PCB-exposed swimming animals were 13 times higher compared to their unexposed controls (Table 1, figure 1b). This difference was significantly different (P ≤ 0.0001). In contrast the PCB-level in muscle of the rest groups (PCB vs. controls) was not significantly different (P ≤ 0.053). Levels in the swimming PCB-exposed animals were 2.7 times higher compared to the resting PCB-exposed group (table 1). This difference was highly significantly different (P ≤ 0.0017). This difference is greater in the unexposed animals, but 6 out of 7 plasma TEQ-levels were below the limit of detection (LOQ). In these cases 50% of the LOQ was used, but this could e.g. result in an overestimation of the TEQ. Plasma TEQ-levels of the swimming PCB-exposed eel were 25 times higher than the background levels of the unexposed eel. In contrast to levels in muscle no significant differences were observed between swimming and non-swimming animals in plasma (Table 1). The ovaria of the swim-Control group had a significantly lower weight than the ovaria of the swim-PCB group (P ≤ 0.001). This explains the significantly lower GSI (P ≤ 0.005) observed in the swim-Control group in comparison to the swim-PCB group The liver weight in the swim-PCB group was significantly lower (P ≤ 0.033) in comparison to the rest-PCB group. This can explain the observed significant difference in H.S.I. between these two groups (P ≤ 0.037).

Blood parameters: Of the measured blood parameters (table 1) a not significantly difference was observed for glucose for the PCB exposed groups: swim groups (PCB vs. CO, P ≤ 0.20), rest groups (PCB vs. CO, P ≤ 0.38). Mean Cortisol levels in the PCB swim and the PCB rest groups were only 30% and 43% of the respective control groups (table 1). However due to a large variance this lowering was not statistically significant (Swim: P ≤ 0.083, Rest: P ≤ 0.33). Plasma-pH, ion levels (sodium and potassium), plasma lactate acid, haemoglobin and hematocrit were unaffected by PCB-exposure.

Oxygen consumption: From 18 days onwards, the PCB-swim group had significantly lower oxygen consumption values than the control swim group (P ≤ 0.05). Hereafter this difference further increased (figure 2, table 1 & 2).

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As the eels did not show any external activities during the oxygen consumption measurements at the end of the protocol, the observed resting rates corresponded to the Standard Metabolic Rate (SMR). For the PCB-swim group the SMR was 25% lower than the control-swim group, a significant difference (table 1, figure 3). The SMR values of the PCB- rest group did not significantly differ (only 7% lower) from those of the control rest-groups. Although the SMR of the PCB-swim group was 20% lower in comparison to the PCB-rest group, this difference was not statistically significantly (P ≤ 0.07) (figure 2). The SMR of the control-swim and control-rest groups did not differ SMR (figure 3). The Cost of Transport (COT, table 3), defined as the amount of oxygen used to transport one kg fish over one km, is significantly lower (P ≤ 0.03) in the PCB group after 100 km and continues up to 800 km (P ≤ 0.0001, table 3). This implies that PCB exposed animals have lower costs of transport.

Covariance analysis on Organ parameters: The ovary weight of the swim groups was significantly higher (P ≤ 0.04) in the PCB exposed group in comparison to the control group. The Analysis of Covariance indicated that this was not due to differences in body weight, but due to PCB exposure (P ≤ 0.008). In contrast, there was no significant difference (P ≤ 0.67) between the rest groups (PCB and control) for ovary weight. The Spleen weight was significantly higher (P ≤ 0.023) in the PCB exposed swim group in comparison to the control swim group. Also for both PCB exposed groups (swim vs rest) this was significantly different (P ≤ 0.019). For the PCB exposed swim group vs the Control-Rest group the results was just significantly different (P ≤ 0.045). The Covariance analysis indicated that this was not an effect due to differences in body weight but a highly significant effect due to PCB exposure (P ≤ 0.008). In contrast there was no significant difference (P ≤ 0.148) between the rest groups (PCB and CO) for spleen weight The liver weight was not significantly different for the swim groups (P ≤ 0.57) nor was for the rest groups (P ≤ 0.36). The Covariance analysis indicated that this was not an effect due to a group effect (PCB exposure). For the eye diameter a Covariance analysis was performed with body length as cofactor. The Covariance analysis indicated that there was not an effect due to a group effect (PCB exposure) (P ≤ 0.83).

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Figure 1: Data from 4 eel groups: control and PCB-exposed eels, either resting or swimming at 0.5 BL/s for 27 days. a) weight loss, b) PCB-levels in muscle c) cortisol level in plasma, d) Standard Metabolic Rate (SMR), e) Spleen somatic Index, f) Gonado somatic Index. *, ** and ***: denotes a significant difference at respectively P ≤ 0.05, P ≤ 0.01; P ≤ 0.001

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Figure 2: Average oxygen consumption profiles of control and PCB-exposed eels swimming at 0.5 BL/s for 27 days (800-km). The oxygen consumption of the PCB-exposed eels is significantly depressed compared to the controls from day 18 onwards. The PCB-group was dosed intraperitoneally with an environmentally relevant mix of 5 mg PCB-153/kg , 7 μg PCB-126/kg and 50 μg PCB-77/kg eel PCB-77

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Table 1: Means ± SD of data obtained from 4 experimental groups of female silver eels: PCB-exposed or unexposed either resting or swimming for 27 days.

PCB-exposed Controls Rest Swim Rest Swim N 6 7 7 6 Eye-Index 10.73 ± 1.73 10.57 ± 2.20 10.03 ± 0.90 10.26 ± 3.00 Liver weight (g) 7.15 ± 1.21 11.38 ± 4.10 ** 8.45 ± 2.54 9.60 ± 2.34 Hepato-somatic index 0.80 ± 0.09 1.19 ± 0.39 ** 0.93 ± 0.32 1.09 ± 0.14 7.72 ± 1.23&&& Ovary weight (g) 11.45 ± 1.69 11.08 ± 2.09 11.45 ± 1.54 *** Gonado-somatic index 1.29 ± 0.17 1.13 ± 0.15 && 1.25 ± 0.16 0.89 ± 0.18 * Spleen-somatic index 0.056 ± 0.0093 0.129 ± 0.063*** 0.07 ± 0.023 0.056 ± 0.019 Eye-index 4.22 ± 0.64 4.24 ± 0.88 4.02 ± 0.29 4.11 ± 1.20 Bodyweight before exposure (g) 941 ± 46 1068 ± 136 991 ± 60 1036 ±150 Total eel length (cm) 74.5 ± 2.1 77.4 ± 2.5 74.8 ± 3.7 73.9 ± 4.1 Swim distance (km) 0 767 ± 112 0 781 ± 124 Bodyweight start swim period (g) 893 ± 88 998 ± 90 895 ± 65 936 ± 127 Bodyweight end swim period (g) 857 ± 99 936 ± 99 845 ± 87 847 ± 103 Weight loss (g) over 27 days 36.1 ± 10.4 62.8 ± 30.9 * 50.1 ± 28.2 89.2 ± 58.4 PCB in muscle (pmol TEQ/g fat) 138 ± 150 381 ± 44 * 4.1 ± 3.7a 31 ± 29 PCB in plasma (pmol TEQ/g fat) 1570 ± 1367 1526 ± 913 63 ± 103 b 64± 75 % fat muscle 25 ± 6.1 27 ± 3.5 34 ± 9.6 27 ± 7.7 % fat plasma 2.4 ± 0.9 1.3 ±0.7 3.4 ± 2.0 1.5 ± 1.1 Standard Metabolic Rate (mg kg-1 h-1) 43.4 ± 6.4 * 54.2 ± 12.4 & 58.2 ± 16.7 58.4 ± 12.4 PH-plasma 7.65 ± 0.07 7.68 ± 0.07 7.65 ± 0.05 7.64 ± 0.06 Sodium (meq/l) 163.3 ± 1.9 161.8 ± 5.5 176.5 ± 22.0 173.3 ± 14.5 Potassium (meq/l) 2.91 ± 0.49 2.62 ± 0.37 2.57 ± 0.76 2.97 ± 0.89 Calcium (mmol/l) 1.43 ± 0.08 1.66 ± 0.24 1.63 ± 0.31 1.54 ± 0.54 Cortisol (ng/ml) 11.20 ± 9.14 7.81 ± 5.99 26.07 ± 28.45 25.87 ± 21.91 Glucose (mM) 5.44 ± 2.01 5.38 ± 1.03 7.13 ± 2.75 8.44 ± 5.52 Lactic Acid (mM) 1.18 ± 0.53 1.13 ± 0.58 1.02 ± 0.35 1.95 ± 1.34 Hemoglobin (mM) 0.33 ± 0.10 0.35± 0.07 0.35 ± 0.03 0.37 ± 0.09 Hematocrit (%) 33.0 ± 3.74 37.6 ± 4.96 37.7 ± 3.51 33.8 ± 4.27

a: 6 of 7 levels were below limit of detection b: 4 of 7 levels were below limit of detection *, ** and ***: denotes a significant difference between the Rest and Swim group at respectively P ≤ 0.05, P ≤ 0.01; P ≤ 0.001 &: &&: and &&&: denotes a significant difference between the PCB and Control group at respectively P ≤ 0.05, P ≤ 0.01; P ≤ 0.001

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Km Control (mean ± stdev) PCB (mean ± stdev) P-value [mg.kg-1.h-1] [mg.kg-1.h-1] 100 73.8 ± 15.9 71.2 ± 15.2 0.42 200 79.5 ± 19.9 75.6 ± 17.6 0.17 300 80.8 ± 19.4 77.0 ± 17.5 0.09 400 81.4 ± 18.7 77.5 ± 17.7 0.04* 500 83.6 ± 19.2 79.2 ± 17.3 0.009** 600 85.9 ± 19.3 81.7 ± 18.2 0.009** 700 87.9 ± 19.1 83.3 ± 19.1 0.003** 800 88.9 ± 19.2 83.3 ± 18.9 0.0001***

Table 2: Mean Oxygen consumption in [mg.kg-1.h-1] in a control swim group and a PCB exposed group related to distance. The oxygen consumption is significantly suppressed after 400 km (which corresponds with 18 days in figure 2) up to 800 km. *, ** and ***: denotes a significant difference between the Conytrol and PCB group at respectively P ≤ 0.05, P ≤ 0.01; P ≤ 0.001

Km COT Control group COT PCB group P-value (mean ± stdev) (mean ± stdev) -1 -1 -1 -1 [mg O2. kg eel . km ] [mg O2. kg eel . km ] 100 56.9 ± 11.3 51.9 ± 10.7 0.03* 200 61.3 ± 14.6 55.1 ± 12.5 0.002*** 300 62.3 ± 14.4 56.0 ± 12.3 0.0001*** 400 62.8 ± 13.6 56.4 ± 12.5 0.0001*** 500 64.6 ± 14.3 57.7 ± 12.4 0.0001*** 600 66.4 ± 14.5 59.6 ± 13.3 0.0001*** 700 68.1 ± 14.5 60.7 ± 14.2 0.0001*** 800 69.0 ± 14.7 60.7 ± 14.0 0.0001***

-1 -1 Table 3: Cost of Transportation values (COT) in [mg O2. kg . km ] in a control swim group and a PCB exposed group related to distance. The COT is already significantly suppressed after 100 km up to 800 km. *, ** and ***: denotes a significant difference between the Conytrol and PCB group at respectively P ≤ 0.05, P ≤ 0.01; P ≤ 0.001

Discussion

General condition The high Eye-Index (table 1) between 10.0-10.7 indicates that all eels were in the silver (migratory) stage. The transition point towards the silver stage is around 6-7 (Pankhurst 1982, Durif 2003). Recently we demonstrated that also yellow eels are able to swim long distances in the swim tunnels (van Ginneken et al. 2005a), which was, however, in fresh water.

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In the experiment described in this study most of the animals were able to swim during the full experimental period of 27 days. However, specially swimming animals experienced non-PCB-related skin problems, sometimes even resulting in death. This problem had occurred in two earlier experiments using marine water (Houtman et al., unpublished results) not-withstanding good water conditions, flumequine-pre-treatment and the application of UV-filters. Fish disease specialists (CDI Lelystad) could not identify infectious diseases. Eels are very plastic with respect to osmoregulation and adapt quickly to both freshwater and seawater environments (Cutler & Cramb 2001). Experiments performed at our laboratory revealed that eels can directly transposed from fresh water into sea water (and vice versa) and that the ionic balance is regained within a few hours (unpublished results prof. Cliff Rankin et al.). In combination with the fact that the animals had been gradually adapted to marine water in 4 weeks and adapted to the swimming tunnels during 2 weeks, the sea-water as such, can not be considered as a stress factor. In addition, the resting animals did not show the same problems. Apparently the combination of the individually non-problematic factors ‘swimming’ and ‘sea water’ is a stress factor for these animals. In this respect recently an important observation was done by Jørgensen et al. (2004). They found for Arctic charr (Salvelinus alpinus) that exposure to PCB impaired smoltification and seawater performance. Fish exposed to 100 mg Aroclor (a PCB mixture) per kg body mass had either a transient or a permanent reduction in plasma growth hormone, insulin-like growth factor-1, thyroxin (T4) and triiodothyronine (T3) during the period of smoltification (Jørgensen et al. 2004). These hormonal alterations corresponded with impaired hypo-osmoregulatory ability in May and June (the smoltification period), as well as reduced growth rate and survival after transference to seawater (Jørgensen et al. 2004). It is possible that the 'silver'-eels (migratory) in this experiment had problems with a continuation of the 'silvering' process (metamorphosis) due to PCB exposure in combination with transition to salt water. Energy use ‘Silvering' is primarily considered as an adaptation of the animal to a long-term migration (Durif et al. 2005). Eels accumulate lipids in the muscle prior to migration, needed for both energy usage and gonad development. Recently we measured in wild Grevelingen eels mean fat percentages of 23.5 % in yellow eel and 27.3% in silver eels (van Ginneken et al. 2005c). In this experiment fat percentages were on average 26%, which is still close to the silver Grevelingen eels.

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In our experiment swimming animals lost about 75% more body weight during the 27 days experimental period than their respective resting controls. This was expected as the energy consumption during swimming is higher than during resting. This was shown for silver eels were we demonstrated during a 10 days swim experiment over 390 km in seawater at 0.5 BL/sec that eels use 40% of their initial fat stores for migration and still have 60% of their initial fat stores remaining available for the developing gonads (van Ginneken & van den Thillart 2000). Recently we demonstrated during a 5,500-km simulated migration of yellow eel in 127 litre Blazka swimtunnels over a period of 173 days that the metabolic rate of swimming animals (at 0.5 Body-length per second) was two times higher compared to resting control animals (van Ginneken et al. 2005a). This is extremely low because active metabolic rate in other fish species can be up to 10-20 times the Standard Metabolic Rate (SMR) (Fry 1971). Interestingly the loss in bodyweight in the PCB-dosed animals was 29% lower than the respective controls (Figure 1a). This is surprising because as an effect of (high) dosages of dioxin-like PCBs the ‘wasting syndrome’ normally results in extreme weight loss in spite of normal appetite. The TCDD-related ‘wasting syndrome’ has been reported in fish (Kleeman et al. 1988). As the animals were not fed during the experiments, the significantly reduced weight loss in the PCB-exposed groups can not be ascribed to increased intake of food but may be the result of PCB effects on intermediary metabolism and metabolic rate. The altered glycocorticoid-mediated action with significantly lowered glucose and cortisol levels in the PCB-exposed animals could be associated with a lowered catabolism of structural protein, a diminished conversion of amino acids in the gluconeogenesis and an altered carbohydrate metabolism. The reduced energy consumption during swimming (Figure 2) and the Standard Metabolic Rate (SMR) and reduced weight loss in the PCB-exposed groups compared to the control groups, could therefore be due to a reduced protein synthesis. It is important to note that these differences in SMR were persistent, as they were measured 2 days after the last swimming activity. -1 -1 The Cost of Transport (COT, [mg O2. kg eel . km ]) (table 3) is significantly lower in PCB exposed animals from 100 km up to 800 km which implies that they have lower costs of transport. In many species PCBs alter thyroid hormone homeostasis. Thyroid hormone is very important for both energy homeostasis and adaptation to higher osmotic values as has e.g. been shown for eels (Leloup and de Luze, 1985) such as occurring under marine conditions. PCBs and their metabolites can interfere via multiple and interactive mechanisms with the thyroid hormone system. Evidence suggests that pure congeners or mixtures of PCBs directly

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interfere with the thyroid gland: with thyroid hormone metabolizing enzymes, such as uridine-diphosphate-glucuronyl transferases (UGTs), iodothyronine deiodinases (IDs), and sulfotransferases (SULTs) in liver and brain, and with the plasma transport system of thyroid hormones in experimental animals and their offspring (Brouwer et al., 1998). Eels have been shown to metabolise PCBs in the liver (De Boer et al. 1994), including PCB 77. The latter has been shown to be converted in OH-metabolites known to mimic thyroid hormone (Murk et al., 1994). In studies with mammals it is demonstrated that PCB exposure stimulates thyroid hormone secretion, which usually has a calorigenic action (French et al. 2001, Pelletier et al. 2002). However, for fish, we recently measured in free moving eels with different thyroid levels (control-, goitrogenic group, elevated T3 and T4 group) the heat production with an accuracy of 0.1 mW by direct calorimetry (van Ginneken et al. 2005d). Our results demonstrated no significant difference in heat production or oxygen consumption between a control group and a group with elevated T3 and T4 levels (van Ginneken et al. 2005d). The reduced energy consumption during swimming (Figure 2) and the Standard Metabolic Rate (SMR) and reduced weight loss in the PCB-exposed groups compared to the control groups, could therefore not be explained by a PCB action via thyroid hormone.

PCB-levels and -effects In this experiment, the PCB-dosage was not very high, only 10x the consumption standard and within environmentally relevant concentrations (De Boer & Hagel 1994). Due to exercise, fat is mobilised containing the PCB's which go from the muscle to the blood compartment. The circulating free fatty acids are transported by the organs to the mitochondria, where they are degradated in the β-oxidation to provide energy. As lipids are particularly oxidised, in the muscles, PCBs will accumulate. Indeed we observed a 2.7 times higher value in the swim PCB group than in the PCB rest group (Figure 1B). So an important conclusion from this study is that PCBs accumulate in the tissues like muscle were fat is burned. This mechanism of PCB accumulation in muscle after exercise is observed in the PCB group as well as in the Control group. In the latter group the accumulation must be due to a relocation of the naturally occurring PCBs. Six of the seven levels in the resting control group were below the limit of detection. In the blood plasma of the PCB-exposure group, lipid levels were the same in the swimming and resting animals and consequently the TEQ-levels were the same (table 1).

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PCBs and immuno-suppression PCB contamination could mean further interference with the eel energy metabolism and thyroid hormone status reducing the chance of the animals to reach their spawning grounds. In addition, toxicants like PCBs have been related to immuno-suppression, reduces resistance to diseases, viruses and parasites. For example, there are observed increased disease prevalence in benthic fish sampled from various coastal areas containing PCB contaminated sediments (reviewed by Vethaak & Reinhalt, 1992). Several field studies have also reported evidence of disrupted immune function in fish sampled from inshore areas known to be contaminated with PCBs and other xenobiotics (Warriner et al., 1988; Weeks et al., 1990; Arkoosh et al., 1991, 1994). We hypothesize that when eels burn fat from poorly perfused storage organs such as the adipose tissue the blood containing PCBs will initially distributed into highly perfused organs such as the liver, gills, kidney, thymus and spleen. In this respect it is important to notice that the most important organs in the immune function are the thymus, spleen and liver. So exposure to PCBs could lead to immuno-suppression,and reduce resistance to diseases caused by viruses and parasites. This has been observed in several studies. E.g. a thymus atropy was recorded in European flounder (Platichthys flesus) exposed to 50 mg PCB-126/kg (Grinwis et al. 2001). It was hypothesised that this may have an impact on the specific resistance against infectious diseases (viral, bacterial, parasitical) in the field situation. Also with respect to the effect of PCBs on the immunologically important spleen (Taysse et al. 1998) contradictory results are found. In some studies a reduction of the spleen weight is observed (Nakata et al. 2002) while in other studies a spleen hypertrophy was observed (Greichus et al. 1975). In our study we observed a significantly increase of the spleen weight. Spleen and head-kidney are immune organs in fish implicated in the integrity of the organism (Taysse et al. 1998). The spleen mainly produces leucocytes (Iwama et al. 1996). The study of Taysse et al. (1998) indicated for carp that these two organs also can contribute to the biotransformation process for the elimination of xenobiotics. Increased biotransformation by the spleen may explain the observed hypertrophy of the spleen in this study.

PCBs and effects on reproduction: An endocrine disrupting effect of PCB's on LH secretion and an inhibition of gonadal growth, resulting in a lower GSI, has been observed in another study (Khan et al. 2001). However the significantly higher GSI (P≤ 0.049) in our PCB-exposed swim group in

184 Chapter 12 comparison with the Control-swim group can possibly be ascribed to the unexpected lower ovary weight in the Control-swim group and not due to a stimulating effect of PCBs eg. working as pseudo-estrogens. When the eel do reach their spawning grounds, elevated PCB- levels could interfere with PCBs pseudo-estrogens sex hormone function, reproduction and hatching success of the eggs. PCBs and other persistent organic pollutants can be passed on from the mother animals to eggs and impair larval survival and development (Gutleb et al., 1999). PCB-effects on impaired larval development are not only mediated by thyroid hormone disruption, but also via disruption of thyroid hormone homeostasis (Brouwer 1991; Zile 1992). Recently an inverse relationship between the TEQ-level and the survival period of fertilised eel eggs has been found (Palstra et al. 2005). This further adds to the suggestion that the current levels of PCBs and other dioxin-like compounds may seriously impair the survival and reproduction potential of the European eel.

Possibly the world-wide decline of eel populations is a multi-causal problem, but exposure to PCB's seem to contribute via at least two mechanisms: impaired survival of the larvae and impaired energy metabolism during migration.

Acknowledgments

The eel migration project at the University Leiden was supported by a grant of the Technology Foundation (subsidized by the Netherlands Organization for Scientific Research) STW-project no. LBI66.4199, by the EU EELREP project no. Q5RS-2001-01836 and by EURO CHLOR (project officer: drs C. de Rooij).

Literature

Arends, RJ, Van der Gaag, R, Martens, GJM, Wendelaar Bonga, SE and Flik, G (1998). Differrential expression of two pro-opiomelanocortin mRNAs during temperature stress in the common carp (Cyprinus carpio L.) J Endocrinol., 159: 85-91.

Arkoosh, M.R., Cassillas, E., Clemons, E., McCain, B. & Varanasi, U. (1991). Suppression of immunological memory in juvenile chinook salmon (Oncorhunchus tshawytscha) from an urban estuary. Fish & Shellfish Immunology, 1: 261-278. Arkoosh, M.R. clemons, E., Myers, M. & Cassillas, E. (1994). suppression of B-cell immunity in juvenile chinook salmon (Oncorhunchus tshawytscha) after exposure to either polycyclic aromatic hydrocarbon or to polychlorinated

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biphenyls. Immunopharmacology and Immunotoxicoloy, 16: 293-314. Brouwer, A., (1991). The role of enzymes in regulating the toxicity of xenobiotics. Role of biotransformation in PCB-induced alterations in vitamin A and thyroid hormone metabolism in laboratory and wildlife species. Biochem. Soc. Trans. 19, 731–738. Brouwer A., D.C. Morse, M.C. Lans, A.G. Schuur, A.J. Murk, E. Klasson-Wehler, A. Bergman, T.J. Visser (1998). Interactions of persistent environmental organohalogens with the thyroid hormone system: mechanisms and possible consequences for animal and human health. In: Toxicology and Industrial Health 14, nos. 1/2 : 59-84. Castonguay, M.; Hodson, P.V.; Moriarty, C.; Drinkwater, K.F.; Jessop, B.M.(1994). Is there a role of ocean environment in American and European eel decline ? Fish.Oceanogr. 3: 197-203. Cutler, C.P.; Cramb, G. (2001). Review: Molecular physiology of osmoregulation in eels and other teleosts: the role of transporter isoforms and gene duplication. Comparative Biochemistry and Physiology A, 130 : 551-564. de Boer, J. & Hagel, P. (1994). Spatial differences and temporal trends of chlorobiphenyls in yellow eel (Anguilla anguilla) from inland waters of the Netherlands. Sci. Total Environ., 141: 155-174. de Boer, J., van der Valk, F., Kerkhoff, M.A.T., Hagel, P. & Brinkman, U.A.Th. (1994). 8-Year study on the elimination of PCBs and other organochlorine compounds from eel (Anguilla anguilla) under natural conditions. Environmental Science and Technology, 28: 2242-2248. Dekker, W. ( 2004). Slipping through our hands. Population dynamics of the European eel, PhD thesis, University of Amsterdam, 186 pp, ISBN 90-74549-10-1. DeVito, MJ, Birnbaum, LS. (1994) Toxicology of dioxins and related chemicals. Dioxins and health. Schecter A (Ed.) Plenum Press, New York, :139-162. Durif, C. (2003). The downstream migration of the European eel Anguilla anguilla: Characterization of migrating silver eels, migration phenomenon, and obstacle avoidance. PhD Thesis, University Paul Sabatier, Toulouse. Durif, C.; Dufour, S.; Elie, P. (2005). The silvering process of Anguilla anguilla: a new classification from the yellow resident to the silver migrating stage. J.Fish.Biol. 66: 1025-1043. French, J.B.Jr; Voltura, M.B.; Tomasi, T.E. (2001). Effects of pre- and postnatal polychlorinated biphenyl exposure on metabolic rate and thyroid hormones of white-footed mice. Environmental Toxicology and Chemistry 20: 1704-1708. Fry, F.E.J. (1971). The effect of environmental factors on physiology of fish. In: Fish Physiology (Edited by Hoar W.S. and Randal D.J.) Vol. VI, p 1-98, Academic Press, New York. van Ginneken, V. and van den Thillart, G.(2000). Eel fat stores are enough to reach the Sargasso, Nature 403: 156-157. van Ginneken, V.; Haenen O.; Coldenhoff, K.; Willemze, R.; Antonissen, E.; van Tulden, P.; Dijkstra, S.; Wagenaar, F.; van den Thillart, G. (2004) Presence of virus infections in eel species from various geographic areas. Bull.Eur.Ass. Fish.Pathol. 24:268-271. van Ginneken, V.; E.Antonissen, U.K.Müller, R.Booms, E.Eding, J.Verreth, G.van den Thillart (2005a). Eel migration to the Sargasso: remarkably high swimming efficiency and low energy costs. J.Exp.Biol., 208: 1329-1335. van Ginneken, V.; B.Ballieux, R. Willemze, K.Coldenhoff, E.Lentjes, E. Antonissen,

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O.Haenen, G.van den Thillart (2005b). Hematology patterns of migrating European eels and the role of EVEX virus. Comp.Biochem.Physiol. C, 140: 97-102. van Ginneken, V.; C.Durif; P.Balm, K.M. Verstegen, Ron Boot, G.van den Thillart (2005c). Silvering of European eel (Anguilla anguilla L.): Seasonal changes of morphological and metabolic parameters. Animal Biology, accepted. Van Ginneken, V.; Ballieux, B.; Antonissen, E.; van der Linden, R.; Gluvers, A.; van den Thillart, G. (2005d). Direct calorimetry of free moving eels with manipulated thyroid status. Naturwissenschaften, accepted. Goldstein, JA & Safe, S. (1989) Mechanism of action and structure-activity relationships for the chlorinated dibenzo-p-dioxins and related compounds. Halogenated biphenyls,terphenyls, naphtalenes, dibenzodioxins and related products. Kimbrough and Jensen (eds) Elsevier Amsterdam 1989. pp. 239-294. Greichus, Y.A.; Call, D.J.; Ammann, B.M. (1975). Physiological effects of polychlorinated biphenyls or a combination of DDT, DDD, and DDE in penned white pelicans. Arch.Environ.Contam.Toxicol. 3: 330-343. Grinwis, G.C.; van den Brandhof, E.J.; Engelsma, M.Y.; Kuiper, R.V.; Vaal, M.A.; Vethaak, A.D.; Wester, P.W.; Vos, J.G. (2001). Toxicity of PCB-126 in European Flounder (Platichthys flesus) with emphasis on histopathology and cytochrome P4501A induction in several organ systems. Arch.Toxicol. 75: 80-87. Gutleb, A.C., Appelman, J., Bronkhorst, M., van den Berg, J.H.J., Spenkelink, A., Brouwer, A., Murk, A.J. (1999): Delayed effects of pre- and early-life time exposure to polychlorinated biphenyls (PCBs) on tadpoles of two amphibian species (Xenopus laevis and Rana temporaria). Environ. Toxicol. Pharmacol. 8: 1-14. Haenen, O.L.M.; Banning van, P.; Dekker, W.(1994). Infection of eel Anguilla anguilla (L.) and smelt Osmerus eperlanus (L.) with Anguillicola crassus (Nematoda, Dracunculoidea) in the Netherlands from 1986 to 1992. Aquaculture 126: 219-229. Iwama, G.; Nakanishi, T.; Hoar, W.; Randall, D. (1996). The Fish Immune System. Organism, Pathogen, and Environment. 380 pages, Academic Press, ISBN: 0-12 -350439-2. Jørgensen, E.H.; Aas-Hansen, Ø; Maule, A.G.; Strand, J.E.T.; Vijayan, M.M. (2004). PCB impairs smoltification and seawater performance in anadromous Arctic charr (Salvelinus alpinus). Comparative Biochemistry and Physiology, C, 138: 203-212. Khan, I.A.; Mathews, S.; Okuzawa, K.; Kagawa, H.; Thomas, P. (2001). Alterations in the GnRH-LH system in relation to gonadal stage and Aroclor 1254 exposure in Atlantic croaker. Comparative Biochemistry and Physiology B 129: 251-259. Kleeman, J.M.; Olson, J.R.; Chen, S.M.; Peterson, R.E. (1988). Species differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and biotransformation in fish. Toxicol. Appl. Pharmacol. 83: 391-401. Knights, B. (2003). A review of the possible impacts of long-term oceanic and climate changes and fishing mortality on recruitment of anguillid eels of the Northern Hemisphere. The Science of the Total Environment 310: 237-244. Leloup J. and de Luze A. (1985). Environmental effects of temperature and salinity on thyroid function in teleost fish. The Endorcine system and the environment, ed. Follett, B.K., Ishii, S. and Chandola, A. (1985). Japan Sci.Soc. Press, p23-32, Tokyo/Springer-verlag, Berlin. Mukerjee, D. (1998) Health impact of polychlorinated dibenzo-p-dioxins: a critical review. J. Air Waste Manag Assoc. 48: 157-165. Murk A, Morse D, Boon J and Brouwer A (1994): In vitro metabolism of 3,3',4,4'

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-tetrachlorobiphenyl in relation to ethoxyresorufin-O-deethylase activity in liver microsomes of some wildlife species and rat. Eur. J. Pharmacol. Environ. Toxicol.Pharmacol. Sect., 270: 253-261. Murk, A.J., Leonards, P.E.G., Hattum, B. van, Luit, R., Weiden, M.E.J. van der. & Smit. M. 1998. Application of biomarkers for exposure and effect of polyhalogeneted aromatic hydrocarbons in naturally exposed European otters (Lutra lutra). Environmental, Toxicology and Pharmacology, 6: 91-102. Nakata, H.; Sakakibara, A.; Kanoh, M.; Kudo, S.; Watanabe, H.; Miyazaki, N.; Asano, Y.; Tanabe S. (2002). Evaluation of mitogen-induced responses in marine mammals and human lymphocytes by in-vitro exposure to butyltins and non-ortho coplanar PCBs. Environmental Pollution 120: 245-253. Palstra, A.P.; van Ginneken, V.J.T.; Murk, A.J.; van den Thillart, G.E.E.J.M. (2005). Are dioxin-like contaminants responsible for the eel (Anguila anguilla) drama ? Naturwissenschaften, accepted. Pankhurst, N.W. (1982). Relation of visual changes to the onset of sexual maturation in the European eel Anguilla anguilla (L.). J.Fish.Biol. 21: 417-428. Pankhurst, N.W. and Lythgoe, J.N. (1983). Changes in vision and olfaction during sexual maturation in the European eel Anguilla anguilla (L.). J.Fish.Biol. 23:229-240. Pelletier, C.; Doucet, E.; Imbeault, P.; Tremblay, A. (2002). Associations between weight loss-induced changes in plasma Organochlorine Concentrations, Serum T3 concentrations, and resting metabolic rate. Toxicological Sciences 67: 46-51. Stone, R (2003). Freshwater eels are slip-sliding away. Science 302: 221-222. Stryer L. (1988). Biochemistry, W.H. Freeman and Company, New York, 1089 pp, ISBN 0-7167-1843-X. Taysse, L.; Chambras, C.; Marionnet, D.; Bosgiraud, C.; Deschaux, P. (1998). Basal level and induction of Cytochrome P450, EROD, UDPGT, and GST activities in Carp (Cyprinus carpio) immune organs (Spleen and Head Kidney). Bull.Environ. Contam.Toxicol. 60:300-305. van den Thillart, G. and van Waarde, A. (1985). Teleosts in hypoxia. Aspects of anaerobic metabolism. Mol.Physiol. 8: 393-409. van den Thillart , G.; van Ginneken V.; Körner F.; Heijmans, R.; van der Linden, R. and Gluvers, A., (2004). Endurance swimming of European eel. J.Fish Biol. 65: 1-7. Vethaak, A.D. & Reinhalt, T. (1992). Fish disease as a monitor for marine pollution: the case of the North Sea. Reviews in Fish and the Biology of Fisheries, 2: 1-32. Voltura, M.B.; French, J.B. Jr. (2000). Efects of dietary polychlorinated biphenyl exposure on energetics of white-footed mouse, Peromyscus Leucopus. Environmental Toxicology and Chemistry 19: 2757-2761. Warriner, J.E., Mathews, E.S. & Weeks, B.A. (1988). Preliminary investigations of the chemiluminescent respons in normal and pollutant-exposed fish. Marine Environmental Research, 24: 281-284. Weeks, B.A., Huggett, R.J., Warriner, J.E. & Methews, E.S. (1990). Macrophage responses of estuarine fish as bioindicators of toxic contamination. In Biomarkers of Environmental Contamination. (McCarthy, J.F. & Shugart, L.R., eds.) pp. 193- 201. Boca Raton, FL: Lewis Publishers. Zile, M.H., 1992. Vitamin A homeostasis endangered by environmental pollutants. Proc. Soc. Exp. Biol. Med. 201: 141–153.

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A 5,500-km swim trial stimulates gonad maturation in the European eel (Anguilla anguilla L.)

V. van Ginneken1*, S. Dufour2, M. Sbaihi2, P. Balm3, K. Noorlander1, M. de Bakker1, J. Doornbos1, E. Antonissen1, I. Mayer4, G. van den Thillart1

1 Integrative Zoology, Institute Biology Leiden (IBL), van der Klaauw Laboratories, P.O.Box 9511, 2300 RA Leiden, The Netherlands. 2 UMR CNRS/MNHN/UPMC Biology of Marine Organisms and Ecosystems, Museum National d’Histoire Naturelle (MNHN),7 Rue Cuvier, 75231 Paris Cedex 05, France. 3 Animal Physiology, Department of Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. 4 Department of Biology, University of Bergen, N-5020 Bergen, Norway.

Keywords: European eel, gonad maturation, spawning migration, swim trial, gonadotropins, estradiol.

*Corresponding Author: Dr.V.J.T.van Ginneken E-mail: [email protected] Tel: +31(0)71-5277492

Submitted to: Gen. Comp. Endocrinol.

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Van Leeuwenhoek reageert op de kritiek van Christiaan Huygens en stelt dat de paling mogelijk tweeslachtig (hermafrodiet) is. Of nu onder de Alen en Palingen geen Mannelijke geslagten zijn, gelijk ik onder vonden hebbe, dat kleijne Dierkens die uijt verscheijde zoorten bestonden, en welke soo op de bladeren van Aelbesse, kersse, Pruijme, en Rooze in groote menigte gevonden werden, die men den naam van Luijsen geeft, na dat ze op de boomen gevonden werden, en welke Dierkens, alle hare lighame met jonge beset zijn, en ijder zijn geslagt voort brengt, zonder dat eenig Mannelijk geslagt, voor mij te bekennen was, nog ook geen de minste verzameling, en hebbe konnen na speuren. Of nu deze Voortelinge mede plaats heeft inde Alen, en Palingen, dan oft ijder Ael en Paling, nog met Mannelijk Zaad zijn versien, en over sulks Armaphroditen zijn, gelijk wij ons inbeelden door de verzameling, die wij komen te zien, dat eenige slakken zoo danig begaaft zijn, dat staat nog te onderzoeken. Dat ik voor desen niet en hebbe geseijt, dat ik in alle de Alen en Palingen, hare Baar-moeders hebbe ontdekt, hebbe ik met voor dagt te rugge gehouden; omdat ik in tijd en wijle, mijn selven daar omtrent beter mogt komen te voldoen. (Antoni van Leeuwenhoek, Brief No. 169 [102], 10 juli 1696).

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A 5,500-km swim trial stimulates gonad maturation in the European eel (Anguilla anguilla L.).

Summary

The catadromous European eel (Anguilla anguilla L.) undertakes a 6,000-km spawning migration from its freshwater habitats to the Sargasso Sea. In large Blazka swim- tunnels of 127 liter, the physiological effect of such a prolonged swimming performance on sexual maturation in adult female eels was investigated. Two groups of eels were placed in swim-tunnels for 173 days, one group was able to swim at 0.5 body lengths/second (Swim group) covering a distance of c. 5,500-km over the experimental period, and one group kept in static (End Control group). A control group was sampled at the start of the experiment in order to determine the initial stage of reproductive development (Initial Control Group). At the end of the swim trial, the maturation parameters 11-ketotestosterone, pituitary levels of LH and plasma levels of estradiol were higher (although not significantly) in the Swim compared to the End Control group. In addition, no significant differences were observed in most measured morphometric and reproductive parameters, including eye-index, gonadosomatic index, hepatosomatic index, and plasma levels of vitellogenin, cortisol and melanophore-stimulating hormone (MSH). Also, pituitary levels of both MSH, and adrenocorticotropic hormone (ACTH) were unaffected. In contrast, the oocyte diameter was found to be significantly higher in the Swim compared to the End Control group. Based on these observations we conclude that a period of prolonged swimming might be a physiological stimulus necessary for the onset of maturation in the European eel.

1. Introduction

The catadromous European eel (Anguilla anguilla) migrates 6,000-km from its freshwater habitats to the Sargasso Sea where it spawns, following which the adults die (Vøllestad 1986; Tesch 2003). One of the mysteries of the life-cycle of the eel is the endocrinological mechanisms controlling gonadal maturation during its prolonged spawning migration. When silver eels start their oceanic migration in the autumn, they show only a limited degree of gonadal development, with a gonadosomatic index (gonad weight/body weight x 100; GSI) of between 1-2. Silver eels maintained in captivity fail to show any further advance in gonadal development.

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In the migratory silver stage of the European eel it has been demonstrated that a pre- pubertal blockage occurs in the hypothalamus-pituitary-gonad (HPG) axis, resulting in the inhibition of sexual maturation (Dufour 1994). At the hypothalamic level, this pre-pubertal blockage is a result of both a deficiency of gonadotropin-releasing hormone (GnRH) coupled with the inhibition of GHT-release by the neuropeptide dopamine (DA). The consequence of this dual blockage is an inhibition of pituitary GTH production, leading to the inhibition of pubertal development in captive silver eels. The endocrinological mechanism(s) by which this dual blockage is naturally abolished in migrating silver eels is not yet clear. Recently the inhibitory control of the LH ovulatory peak in eel by dopamine (DA) could be abolished by triple treatment with testosterone (T), GnRH agonist (GnRHa) and DA-receptor antagonist (pimozide) (Vidal et al. 2004). The question however remains what is the environmental stimulus to abolish the prepubertal blockage and trigger the gonad maturation of European eel. The main aim of the work presented in this manuscript was to study the effect of exercise on maturation of the European eel. For European eel high FSH-β mRNA expression was detected in females at the pre- vitellogenic stage while very low or no LH-β mRNA expression was detected at this stage (Degani et al. 2003). For Japanese eels similar results were found. In young and pre- vitellogenic fish high FSH-β mRNA expression was observed while in late vitellogenic and maturing fish LH-β mRNA was expressed by (Suetake et al. 2002). Also in ovulating animals LH-β mRNA was expressed (Yoshiura et al. 1999). The mRNA expression profiles of females of European eel, where FSH is expressed prior to LH, suggest that FSH regulates gametogenesis and vitellogenesis while LH, on the other hand, is involved in oocyte maturation and ovulation (references: vide Degani et al. 2003). This pattern of expression where FSH is expressed during vitellogenesis and LH during maturation resembles the pattern of salmonids (Gomez et al. 1999, Degani et al. 2003). However recent results indicate that the matter is more complicated for wild European eels. Indeed, first of all, at least in the wild European silver eel, it is observed that LH is perfectly detectable in the pituitary, (by RIA) and by mRNA (assay and in situ hybridization). So, LH is detectable as well as FSH (Schmitz et al.,2005). Second, a significant increase in pituitary LH content is observed at silvering (Marchelidon et al., 1999 ; Sbaihi et al., 2001; Durif et al., 2005). So it is not a situation of “all or nothing “ between LH and FSH in the silver eel.

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In explaining the control of steroids via the HPG-axis and its negative feedback control a clear correlation was demonstrated between steroids and gonadotropin expression and its negative feed-back loops in eels. For the European eel, Querat et al. (1990) found a strong positive effect of estradiol treatment on the level of putative LH-β mRNA in European eel, but did not measure FSH-β. But the results of the study of Schmitz et al. (2005) are indicative for an opposite regulation of LH ß (increase) and FSH.ß (decrease). For the Japanese eel, injected with salmon pituitary homogenate, it was observed that LH-β gene expression correlated with serum estradiol-17β and testosterone levels during oocyte maturation (Nagae et al. 1996, Suetake et al. 2002). The data from the Paris research group show during experimental maturation, and under the feedback of steroids, that there is an opposite regulation of the expression of LH and FSH (increase in LH and decrease in FSH) (Schmitz et al., 2005). So, it is clear that LH will accumulate in the pituitary throughout vitellogenesis and be ready for triggering the final steps (oocyte maturation and ovulation). But it is still not clear whether FSH alone controls the entire vitellogenic process. To justify the measurement of LH in the present study and examine the natural trigger for maturation we aimed at comparing the changes in various pituitary hormones under the effect of the swimming challenge. We choose to measure ACTH (as representative of the control of stress and metabolism), and LH as representative of a gonadatropin hormone. Also we measured αMSH in plasma and the pituitary. The rationale behind this is that ACTH and αMSH are the products of one pituitary hormone precursor, proopiomelanocortin (POMC). A distinctive feature of POMC, is the presence of multiple melanocortin core sequences, and one copy of the opiod, β-endorphin (Alrubaian et al. 2003). For teleost there are two melanocortin regions in POMC: ACTH/ αMSH, β-MSH while in contrast to certain extant lobe-finned fish (lungfish) and the tetrapods γ-MSH is lacking (Alrubian et al. 2003). A model for the interaction between ACTH/cortisol and the HPG axis can be via the Steroidogenic acute regulatory protein (StAR). This is a key molecule for steroid production by translocating cholesterol from the outer to inner mitochondrial membrane (Li & Takei 2003, see further discussion).

Following the HPG-axis model, E2 is the main inducer of hepatic VG synthesis.

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The presence of vitellogenin granules mark the beginning of ‘exo-vitellogenesis’ which is known to be the result of FSH secretion (Degani et al. 2003). However for European eel the respective roles of FSH and LH in the early steps of vitellogenesis in the eel may not be as clearly distinct, specially as silvering is marked by a significant increase in LH (Marchelidon et al.,1999; Sbaihi et al., 2001, Durif et al., 2005). Also, recent work of Schmitz et al. (2005) demonstrated that both LHβ and FSHβ are submitted to an opposite regulation (increase in LH and decrease in FSH) during experimental maturation (gonadotropic treatment) or under steroid treatments. Although no adult eel has ever been caught in the Sargasso Sea to determine the GSI at spawning, based on observations from hormone-treated eels it has been concluded that fully mature adults attain a GSI value of between 40-70 (references vide van Ginneken et al. 2005d). In view of the pronounced pre-pubertal blockage evident in captive silver eels it has been proposed that the unique environmental factors experienced by the eel during its 6,000- km spawning migration may, in some way, interact upon the endogenous neuroendocrine mechanisms controlling sexual maturation. These environmental factors include temperature (Boëtius & Boëtius 1967), light, salinity (Nilsson et al. 1981) and water pressure (Fontaine 1993). The latter factor is based on one observation of a migrating eel with swollen belly at the Bahamas at 2,000-m depth (Robins et al. 1979). The influence of temperature, light, and salinity on maturation in silver eels have been studied, with no clear effect on the HPG-axis (Boetius & Boetius 1967, Nilsson et al. 1981). As it has been assumed that maturing eels migrate at considerable ocean depths, water pressure has been investigated in the laboratory (Sebert & Barthelemy 1985, Simon et al. 1988) and under simulated conditions in the field (Dufour & Fontaine 1985). In laboratory studies using pressure chambers, exposing eels to high hydrostatic pressures of either 2.5 Mpa (Nilsson et al. 1981) or 101 atmospheres (Sebert & Barthelemy 1985, Simon et al. 1988) had no effect on gonadal maturation, although changes in metabolism were observed. This was even the case following long-term exposure to high pressure for a period of one month (Simon et al. 1988) or 4 months (Nilsson et al. 1981). In contrast, a pronounced stimulation on the HPG-axis was recorded in a field study, where female silver eels were kept in cages at a depth of 450 m in the Mediterranean Sea (Dufour & Fontaine, 1985). In this study, eels kept for 3 months at this depth showed a slight increase in gonad development (GSI = 2.3) compared to the controls (GSI = 1.6). However, most strikingly, the pituitary GTH content in the eels kept at depth was 27-times higher than in the controls (Dufour & Fontaine 1985).

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Remarkably, exercise has never been investigated as a potential stimulating factor. Major physiological and endocrinological changes are known to occur as a result of prolonged exercise in catadromic and anadromic fish species (Smith 1985). As European eels are assumed to swim 5,000-6,000-km over a period of 6 months to their spawning grounds (Tesch 2003), we hypothesized that the physiological demands of this prolonged migration may influence the mechanisms controlling sexual maturation. To that end, using experimental swim-tunnels, we investigated the effect on prolonged swimming exercise on adult female silver eels. Changes in morphometric and physiological parameters associated with sexual maturation was investigated in both eels allowed to swim for 6-months and in resting eels. In particular, the effect of prolonged swimming on parameters of the hypothalamus-pituitary- gonad (HPG) axis and ACTH-cortisol axis were investigated.

2. Material and Methods

2.1 Rationale of the experiment, selection of the animals and experimental conditions It was initially our intension to simulate the 5,500-km migration of European eel (Anguilla anguilla L.) to the Sargasso Sea in 22 Blazka swim tunnels with approximately 20 years old silver eels caught during their spawning migration at a low temperature of 5-7 º C in sea water mimicking the deep-sea situation. However, for three consecutive years we were unable to perform these experiments because the animals caught in the Grevelingen (the Netherlands) during their seaward migration showed anaemia, blood in the abdominal fluid and hemorrhage all over the body during a simulated migration in sea-water in the swim tunnels in the laboratory. Consequently, the animals stopped swimming after 1,000-1,500 km (van Ginneken et al. 2005a). Finally we detected EVEX (Eel Virus European X) a rhabdovirus in our animals (van Ginneken et al. 2005a). In a survey conducted in eel species from various geographic regions we found that viruses mainly EVEX, HVA (Herpesvirus anguillae) and EVE (Eel Virus European) were commonly present among eel populations (van Ginneken et al. 2005b). Therefore we choose to perform our endurance swimming experiments with yellow eels from an eel farm in fresh water at 18º C. Eel farmers immunize their eels against eel viruses by means of bath exposure at the glass eel (elver) stage to adult, virus positive eels (van Ginneken et al. 2005a). With these animals we were able to make a simulated migration of 5,500-km and calculate the energy costs of migration over this distance (van Ginneken et al. 2005c).

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In addition, we observed among these yellow eel unexpectedly, an initially and significant signs of maturation which we found interesting and worth to mention in this manuscript.

2.2. Animals and husbandry conditions Three year old hatchery-reared eels were used, having a mean length and weight of 71.4 ± 4.2 cm and 792 ± 104 gms respectively. These eels were known to be all females as under hatchery conditions all males of similar age are considerably smaller (Tesch 2003). They were kept in fresh water for a period of one month before being used in the experiment. The recirculation system and swim tunnels were placed in a climatized room with a constant temperature of 18 °± 0.3 °C. The water temperature was kept at 18 °± 0.1 °C, and the animals

were kept under conditions of constant dark conditions. The NH3 and NO value of the water

was measured daily. At values >0.1 ppm NH3 the water was refreshed from a 3000 l tank elsewhere (van den Thillart et al. 2004).

2.3. Blazka swim-tunnels The Blazka swim-tunnels were calibrated with a Laser Doppler technique at the Delft Hydraulics Laboratory, Technical University Delft. The dimensions of each swim-tunnel was: length of 200.0 cm, diameter of the outer and inner swim-tunnel tubes were 28.8 19.0 cm respectively, and having a volume of 127.1 ± 0.9 liters (n=5). The power of the engine is 400 watt while the propeller consists of three 7.5 inch blades with a pitch of 7 inches. At the top end of the swim- tunnel is placed a PVC flow conditioner with a length of 50 cm while at the propeller-end a flow conditioner of 12 cm length is placed. At the bottom-end of the tunnel a screen is placed of plaited silver wire of 1-mm thickness to conduct the electrical current used for the stimulation of the fish to swim. The electrical current was sinusoid, with a peak of 6.5 V, a frequency of 1 s and a duration of c. 20 ms. This was applied via a central generator in a resuming way, sequential, to all 22 swim tunnels. The stimulation was only used initially when the eels had to learn to swim in the tunnels; in most cases this was not required. After several days the grids were not used any more. (van den Thillart et al. 2004).

2.4. Experimental design The female eels were randomly divided between three experimental groups: a Swim group (n=9), and an End-Control group (n=6), and an Initial Control group (N=11).

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The Initial Control group was sampled at the start of the experiment to describe the initial maturational status of the animals. The remaining 15 eels were placed in individual 127 litre Blazka swim-tunnels. For the Swim group, depending on the size of each individual, the swimming speed of the water in the swim tunnels was set at 0.5 body length per second. This water speed equated to the eels swimming c. 5500 km over the experimental period of 173 days, closely mimicking the exercise the eel performs during its natural migration (Ellerby et al. 2001). The End-Control group experienced the same water quality conditions but with no water current. All fish were not fed during the experimental period.

2.5. Sampling procedure Animals were quickly anaesthetized with 300 PPM MS222 (3-aminobenzoic-acid-ethyl- ester methanesulfonate salt, Sigma, St. Louis, USA). After three minutes the anaesthetized fish were taken out of the swim-tunnel and blood was collected with a heparinized syringe (flushed with 3000 units heparin per ml blood). The fish were humanely killed by decapitation, following which the alimentary tract, heart, and liver were dissected out and weighed. The gonads were then excised, and after weighing to determine the GSI, a mid- portion of the gonad was fixed in Bouin’s fixative. Finally, the pituitary was quickly dissected out taken out and directly homogenized in 0.3 % NaCl with an ultrasonic thorax and stored at -80oC pending analysis. In the pituitary LH, ACTH and MSH were determined with RIA’s (see further). Blood was directly centrifuged at 10,000 rpm for 5 mins, and the plasma divided in Eppendorf tubes (25 μl, 50 μl, 100 μl, 100 μl and 100 μl) for measurement of cortisol, vitellogenin (VTG), 11-ketotestosterone (11-kT), 17-β-estradiol (E2) and melanophore-stimulating hormone (MSH) respectively, and stored at -80oC pending analy- sis.

2.6. Analytical methods 2.6.1. Hormone analysis Cortisol was measured by radioimmunoassay at Nijmegen University according to the protocol of Balm et al. (1994). VTG was measured by immunoenzymatic assay (ELISA) (Burzawa-Gerard & Dumas-Vidal 1991) at MHNH in Paris. The LH content in the pituitary homogenate was also determined at MHNH in Paris by radioimmunoassay following the protocol of Dufour et al. (1983). The pituitary MSH and pituitary ACTH content were determined by RIA’s as described elsewhere (Balm & Pottinger 1995).

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Plasma levels of 11-kT and E2 were measured specific by radioimmunoassay at Stockholm University, Sweden, as previously described by Sbaihi (2001). Plasma ACTH and α-MSH were analyzed using methodology described in Balm & Pottinger (1995). 2.6.2. Histological analysis After fixation in Bouin’s fixative, gonads were dehydrated in a graded ethanol series and embedded in HistoResin according to standard procedures (Romeis 1968). They were sectioned at 5 μm and stained with haematoxylin and eosine. Per section the length and width of thirty oocytes were measured and then averaged.

2.7. Statistical analysis The condition factor (CF) was calculated according to the equation CF = 100 x W x L-3. For all three groups, Swim- End-Control-, and Initial-Control group, the mean value of all measured parameters was compared pair-wise. A Kruskal-Wallis test was performed on the data to see whether a significant difference was present between the three groups. Normality

of the data and homogeneity of variances were checked by Kolmogorov-Smirnov and Fmax tests, respectively. When data were normally distributed a one-way ANOVA was used with a Bonferroni-correction. Not normally distributed data were tested using a Mann-Whitney U test applying a manual Bonferroni-correction. In order to test if those maturation parameters that were found to be significantly different between the groups where influenced by initial length or weight of the fish, a covariance analysis was performed in SPS6 using a General Linear Model.

3. Results

The eels swam 5533 ± 354 km over a period of 173 days (for details see van Ginneken et al. 2005c). Mean (± sd) values of all measured morphometric parameters between the three experimental groups are shown in Table 1. There were no significant differences in length between the experimental groups at the start of the experiment. However for weight the Swim-groups were somewhat heavier. Therefore we applied a covariance analysis (Table 2). The P-values for the covariance analysis between initial body length and body weight with respectively LH, E2 and oocyte-diameter of the pooled experimental groups gave, (with the

exception of body-weight with E2 : P≤ 0.014), no significant difference between subject factors and dependent variables.

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This analysis indicates that the differences in the maturational parameters GTH-II, E2 and oocyte-diameter observed between the Swim and End-Control groups at the start of the experiment were not due to differences in initial body length or weight. A significant difference was observed between the Swim- and End-Control groups in final body weight at the end of the period of 6 months. In the Swim-group the mean body weight decreased by 20% in the Swim group compared to 10% in the End-Control group at the end of the 6-month swim trial. No significant differences were found in the other measured morphometric parameters between the three groups, including eye-index, gonad- weight, GSI, liver-weight, HSI, heart-weight and heart-somatic-index. Mean values of the measured maturational parameters for the three experimental groups are shown in Table 3. For most measured parameters, including plasma VTG, cortisol, 11kT ACTH and MSH levels, and pituitary levels of MSH and ACTH, no significant differences were recorded between the different groups. Only for the maturation parameters 11kT,

pituitary-LH and 17β-estradiol (E2) measured values were higher (not significantly) in the Swim-group in comparison to the End-Control--group. This can be ascribed to the fact that some animals were ‘responding’ to the treatment while others not. For the different hormones we found the following values of ‘Swim’-animals with an increased level: Cortisol: 5 animals between 86-137 ng/ml, 4 animals between 32-65 ng/ml; Pituitary-LH: one animal 65 ng/pituitary, 4 animals between 7.1-10.3 ng/pituitary; 4 animals between 1.0-5.8 ng/pituitary; estradiol: one animal 7.1 ng/ml, all other animals between 3.3-5.9 ng/ml; 11-kT: one animal 18.4 ng/ml, all other animals between 2.4-5.8 ng/ml; oocyte diameter: 4 animals between 0.17-0.19 mm, 5 animals between 0.14-0.16 mm.

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PARAMETER Swim group End Control Initial Control Kruskal- P-value P-value P-value Wallis (N=9) (N=6) (N=11) SwimÙEnd SwimÙInitial End Control Control Control ÙInitial Control Length (cm) 74.7 ± 3.4 70.6 ± 3.74 71.2 ± 3.9 P ≤ 0.042 AN P ≤ 0.031* P ≤ 0.023* P ≤ 0.797 Initial-weight (g) 914.7 ± 58.37 778.2 ± 66.08 795.0 ± 71.92 P ≤ 0.001 AN P ≤0.001** P ≤0.001** P ≤0.676 Condition-factor 0.221 ± 0.03 0.221 ± 0.02 0.214 ± 0.02 P ≤ 0.896 AN End-weight (g) 734.33 ± 44.86 698.33 ± 60.39 - P ≤ 0.115 AN Weight-difference (in %) -20.58 ± 4.37 -10.26 ± 1.37 - P ≤ 0.000 AN P ≤ 0.000** Eye-index 7.43 ± 1.86 7.32 ± 1.44 6.06 ± 1.15 P ≤ 0.102 AN Gonad-weight (g) 7.51 ± 1.9 8.52 ± 1.58 7.28 ± 2.60 P ≤ 0.363 AN G.S.I. 1.05 ± 0.23 1.22 ± 0.19 1.06 ± 0.31 P ≤ 0.292 AN Liver-weight (g) 5.42 ± 0.88 5.12 ± 1.20 4.91 ± 1.36 P ≤ 0.294 AN H.S.I. 0.72 ± 0.09 0.73 ± 0.12 0.75 ± 0.18 P ≤ 0.722 AN Heart (g) 1.0 ± 0.26 1.13 ± 0.14 1.07 ± 0.19 P ≤ 0.379 AN Heart-Somatic-Index 0.13 ± 0.04 0.16 ± 0.02 0.17 ± 0.03 P ≤ 0.227 AN

Table 1: Morphometric, parameters of female European eel (Anguilla anguilla L.) exposed to endurance swimming. Swim-group: sampled after swimming during 173 days over 5,500-km; Initial Control group sampled at the start of the experiment; End Control group sampled after a resting period of 173 days. Mean ± SD is given. AN: ANOVA, MW: Mann-Whitney U test with Bonferroni correction. *: denotes significant difference P ≤ 0.05; **: denotes significant difference P ≤ 0.001.

LH 17β-estradiol Oocyte-diameter

Length 0.670 0.067 0.581 Body-weight 0.150 0.014* 0.242

Table 2: P-values of the covariance analysis between subject factors (length and Body-weight) versus dependent variables: LH, estradiol and oocyte-diameter for the pooled three experimental groups*, significant difference at the p ≤ 0.05 level.

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PARAMETER Swim group End Control InitialControl Kruskal-Wallis P-value P-value P-value (N=9 ) (N=6 ) (N=11 ) SwimÙEnd SwimÙInitial End Control Control Control ÙInitial Control Vitellogenin 148.0 ± 56.9 Below detection 184.53 ± 189.08 P ≤ 0.751 MW (μg/ml) limit <130.0 11-ketotestosterone 4.89 ± 4.86 2.68 ± 0.46 2.52 ± 0.71 P ≤ 0.131 AN (ng/ml) Cortisol (ng/ml) 80.5 ± 38.8 139.2 ± 62.5 63.8 ± 31.7 P ≤ 0.025* AN P ≤ 0.035* P ≤ 0.280 P ≤ 0.003** Pituitary-ACTH 48.96 ± 22.43 54.3 ± 12.63 48.9 ± 10.06 P ≤ 0.485 AN (ng/pituitary) Plasma –ACTH 146.8 ± 98.49 130.67 ± 57.28 164.33 ± 96.37 P ≤ 0.841 AN (pg/ml) Pituitary-α-MSH 877 ± 833 1072 ± 620 928 ± 671 P ≤ 0.448 AN (ng/pituitary) Plasma-α-MSH (pg/ml) 521.6 ± 239.1 631.8 ± 256.3 359.2 ± 129.3 P ≤ 0.049* AN P ≤ 0.399 P ≤ 0.056 P ≤ 0.008**

Table 3: Endocrinological and maturation parameters of female European eel (Anguilla anguilla L.) exposed to an endurance swimming. Swim- group: sampled after swimming during 173 days over 5,500-km; Initial Control group sampled at the start of the experiment; End Control-group sampled after a resting period of 173 days. Mean ± SD is given AN: ANOVA, MW: Mann-Whitney U test with Bonferroni correction. . *: denotes significantly difference P ≤ 0.05; **: denotes significant difference P ≤ 0.001.

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No correlations were found between the different hormones (elevated levels of certain hormones didn’t indicate that all hormones were elevated for the same animal). It is interesting to notice that the response of the whole swim group in oocyte diameter was rather uniform with enlarged oocytes with increased number of secondary lipid vesicles. This explains why significant differences were recorded in oocyte-diameter between the End- Control- and Swim- group (Fig. 1). The histological appearance of an ovary from a female from both a End-Control and Swim group is shown in figure 2. The ovary from the Swim group clearly shows a higher proportion of secondary oocytes with numerous secondary lipid vesicles in the cortex.

Figure 1: Effect of a swim-trial of 5,500-km on the following maturation parameters: a) pituitary LH, b) plasma E2 levels and c) oocyte diameter. For the three groups: Initial Control (n=11), End- Control (n=6) and Swim group (n=9) the mean ± SD values given. *: denotes significantly difference P ≤ 0.05; **: denotes significantly difference P ≤ 0.01; ***: denotes significantly difference P ≤ 0.001.

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Figure 2: Transverse section of an ovary from a female eel from the a) End-Control- and b) Swim-group. 5 μm HistoResin sections, stained with haematoxylin and eosine.

4. DISCUSSION

During its long 6-month spawning migration, the European eel undergoes pronounced morphological as well as physiological changes. In this study, using experimental swim- tunnels, the effect of constant swimming over a 6-month period on both morphometric and physiological parameters was studied in female yellow eels. In particular, the effect of prolonged swimming performance on the hormones of the HPG-axis and the ACTH-cortisol axis was investigated by radioimmunoassay. We choose to use radioimmunoassays in the present pilot study in order to compare various pituitary hormones in the same individual pituitary extracts. To date, it is still unclear whether sexual maturation is accompanied by increased corticosteroid levels in teleost fishes (reviewed by Idler & Truscott 1972, Pickering 1989). Impaired reproductive performance is a common phenomenon in fish (Donaldson 1990, Barton & Iwama 1991) and other vertebrates following exposure to stress (Moberg 1985). On the other hand, a stimulating effect of cortisol on pituitary LH production in the eel was recently demonstrated both in vitro using pituitary cell cultures as well as in vivo by Huang et al. (1999). Several studies have reported on the negative effect of elevated cortisol levels on the HPG-axis during sexual maturation. For example, in the rainbow trout (Oncorhynchus mykiss) increased cortisol levels has been shown to suppress pituitary GTH release (Carragher et al. 1989) and also to inhibit ovarian steroidogenesis both in vitro (Carragher and Sumpter 1990) and in vivo (Pankhurst and van der Kraak 2000).

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Also, cortisol administration resulted in decreased plasma estradiol-binding capacity in immature female rainbow trout, resulting in the decreased effectiveness of estradiol (Pottinger & Pickering 1990). Conversely, a few studies have reported a stimulatory effect of cortisol on the HPG-axis. For example, Huang et al. (1999) recently demonstrated in pituitary cell cultures as well as in vivo, a stimulating effect of cortisol on pituitary LH- production in the European eel. A model for the interaction between ACTH/cortisol and the HPG axis can be via the Steroidogenic acute regulatory protein (StAR). This is a key molecule for steroid production by translocating cholesterol from the outer to inner mitochondrial membrane (Li & Takei 2003). It is known for mammals that the conversion of cholesterol to pregnenolone, catalyzed by cytochrome P450 side chain cleavage enzyme (P450scc), is the rate-limiting step in steroidogenesis and that StAR is required for this process (Stocco 2001). In general StAR is principally expressed in steroidogenic tissues (Sugawara et al. 1995, Bauer et al. 2000, Kusakabe et al. 2002). For Japanese eel it was demonstrated that the distribution of StAR was indeed in steroidogenic tissues like head kidney and gonads, and to minor extent in brain (Li & Takei 2003). For teleost there are two indication that acute stress can have its impact on steroid production via StAR. For rainbow trout it was demonstrated that acute stress increased StAR transcripts in the head kidney (Kusakabe et al. 2002). Furthermore, for Anguilla japonica, it was demonstrated that ACTH injection elevated both plasma cortisol and StAR mRNA levels in the head kidney 1.5 and 4.5 h after injection (Li & Takei 2003). So these literature data demonstrate that an interaction via both axis (ACTH/cortisol axis and HPG-axis) is possible via StAR. Therefore in this study the effect of prolonged swimming on parameters of the hypothalamus-pituitary-gonad (HPG) axis and ACTH-cortisol axis were investigated. In the present study, plasma cortisol levels measured in the eels of all three experimental groups were in general agreement to those levels reported by van Ginneken et al. (2005e) on the seasonal changes (April-November) in hormone levels in wild eels. In this study, female silver eels had mean plasma cortisol levels of c. 81 ng/ml, while in the present study the Swim- and End-Control groups had mean levels of 81 and 139 ng/ml respectively. This suggests that the stimulation of maturational parameters in the swimming eels were not due to elevated cortisol levels, or an activation of the ACTH-cortisol axis per se. The MSH regions in the Pars Intermedia of the pituitary are the fastest growing regions in the stages from elvers to 12-14 cm yellow eels. (Grandi et al. 2003).

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Therefore it can be concluded that the plasma level of α-MSH increases with prolonged aging of the yellow eels in both Swim- and End-Control group after a period of 173 days as observed in this study. Physiologically, 11-kT is the most important androgen in male teleost fishes, and has been shown to be the major androgen involved in spermatogenesis in the Japanese eel, A. japonica (Miura et al. 1991 a,b, Miura et al. 2003). Despite the fact that 11-kT is generally a male-specific androgen, elevated levels of this androgen has recently been reported in wild New Zealand female eels, A. dieffenbachia and A. australis (Lokman et al. 1998). In teleost fishes, as in other vertebrates, some androgens can be converted to estrogens by the enzyme complex called aromatase, located mostly in the brain (Timmers et al. 1987). However, 11kT is a teleost specific non-aromatizable androgen and for this reason was assumed not to be involved in female reproductive development, including oogenesis. While not involved in the control of oogenesis, Lokman et al. (1998) suggested that 11-kT may play a role in preparing maturing animals for their spawning migration. Indeed, in some studies (Rohr et al. 2001, Lokman et al. 2003) it has recently demonstrated that 11-kT administration induced silvering- related changes in immature A.australis eels, including change in head shape and pectoral fin appearance, structural changes of the skin, and an increase in liver weight and eye index, larger ovaries and more advanced oocytes compared to controls. In our study, 11-kT was slightly, but not significantly, elevated in the Swim group. Compared to the End-Control group, the Swim group showed no significant changes in Eye- index, gonad-weight, G.S.I., liver-weight, H.S.I. heart-weight and Heart-somatic index. In our study at least, this suggests that 11-kT is not directly involved in the physiological changes associated with sexual maturation in female eels during their prolonged spawning migration. However, a possible role in the control of maturation in male eels cannot be excluded. Probably, as elegantly demonstrated in the study of Rohr et al. (2001), the role of 11-kT is limited to the pre-spawning adaptation process, called ‘silvering’. It has now been demonstrated that a pre-pubertal blockage occurs in the HPG-axis in silver eels maintained in captivity, resulting in the inhibition of sexual maturation (Dufour 1994, Dufour et al 2003). It has been suggested that the unique environmental factors experienced by the eel during its prolonged spawning migration may influence reproductive development. Although not conclusive, evidence suggests that hydrostatic pressure can influence gonadal development, including stimulating GTH release in the eel (Dufour & Fontaine 1985).

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In the present study, we investigated whether the physiological demands associated with long-term swimming over a distance of 5,500-km can also have an influence on sexual maturation. Our results show that female yellow eels allowed to swim constantly for almost six months did indeed show early signs of sexual maturation. Compared to the End-Control group, eels in the Swim group showed (not-significantly), higher levels of pituitary LH and

plasma E2. Further, although there was no difference in GSI between the groups, mean oocyte diameter was significantly greater in eels from the Swim group. Most noticeably, a large proportion of oocytes from eels in the Swim group contained numerous lipid vesicles throughout the cortex. The still low GSI value is a clear indication that exogenous vitellogenesis, the principal event responsible for the enormous growth of the oocytes (Nagahama 1994), had not started. This is supported by the low plasma VTG levels measured in all eels. In conclusion, the results indicate that prolonged swimming had the effect of stimulating the HPG-axis resulting in the start of oogenesis, although at a very early, pre- vitellogenic stage. Information on the gonadal development of migrating eels is very scarce, as only a few maturing silver eels have been caught in the open oceans. In the literature, only two cases have been reported where European eels have been caught during their spawning migration to the Sargasso Sea. One female eel was caught off the Faeroe Islands and had a GSI of 2.9 (Ernst, 1975), while another was caught off the Azores and had a GSI of 9.8 (Bast and Klinkhardt, 1988). Although these GSI values are higher than in the Swim-group eels, they are considerably lower than the values observed for hormone-treated sexually mature females (van Ginneken et al. 2005d). This indicates that the eels mature progressively during their spawning migration. In this study we used 22 swim tunnels of 127 liter, which can only keep one adult female eel per tunnel. The advantage of this approach was that every eel individually could be followed. Because every tunnel was supplied with an oxygen electrode (van den Thillart et al. 2004) we could perform refined measurements and calculate per individual animal the energy costs of migration over 5,500-km (van Ginneken et al. 2005c). However, it is our impression (inter alia this study) that eels show a large individual variation in physiological and endocrinological parameters. This was also observed in other work performed in the Leiden research group with hormone treated animals were a large variation was observed in the maturation response of animals injected with carp-pituitary extract (van Ginneken et al. 2005d, Palstra et al. 2005).

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Therefore experiments need to be performed with larger groups of animals. It is our intention in future studies to perform experiments with large groups of silver eels in a stream gutter. This approach can have several advantages: a) pheromones and social interaction (van Ginneken et al. 2005d) of animals swimming in a group may possibly trigger gonad maturation, b) environmental conditions like a low water temperature, salinity and water quality can be better controlled, c) possibly stressful conditions like the limited space of the 127 liter swim-tunnel and the screen with electrical stimulation placed at the end of the tunnel for control of continuous swimming is probably not necessary because silver eels already have the drive for endurance swimming and migration. Furthermore, at the moment there is no RIA available for European eel FSH. But, future studies, using mRNA measurements, will aim at comparing the changes in LHß and FSHß expression after swimming challenge. Also in future studies we should aim at investigating the comparative changes in LH and FSH expression now that the molecular tools have been developed (Schmitz et al., 2005). In conclusion, using experimental swim-tunnels, we have demonstrated that forcing female yellow eels to swim for a distance of over 5,500 km has the effect of stimulating the HPG-axis resulting in the start of oogenesis. These results suggest that a prolonged period of swimming may be a necessary physiological cue stimulating the initiation of sexual maturation in the European eel.

ACKNOWLEDGMENTS

Prof. Dr. M. Richardson is thanked for critically reading the manuscript and for correcting the English. This study was supported by the Netherlands Organisation for Scientific Research (STW-project no. LBI66.4199) and by the European Commission (Project QLRT- 2000-01836).

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Idler, D.R., Truscott, B., 1972. Corticosteroids in fish. In: Steroids in Non-mammalian Vertebrates (ed. D.R. Idler) pp. 127-252. Academic Press, New York. Kusakabe, M., Todo, T., McQuillan, H.J., Goetz, F.W., Young, G. 2002. Characterization and expression of steroidogenic acute regulaqtory protein and MLN64 cDNAs in trout. Endocrinology 143, 2062-2070. Li, Y-Y., Inoue, K., Takei, Y. 2003. Steroidogenic acute regulatory protein in eels: cDNA cloning and effects of ACTH and seawater transfer on its mRNA expression. Zoological Science 20, 211-219.

Lokman, P.M., Vermeulen, G.J., Lambert, J.G.D., Young, G., 1998. Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels (Anguilla dieffenbachi and A. australis) before and at the onset of the natural spawning migration. I. Females. Fish Physiol. Biochem. 19, 325-338. Lokman, P.M., Rohr, D.H., Davie, P.S., Young, G. 2003. The physiology of silvering in Anguillid eels: Androgens and control of metamorphosis from the yellow to silver stage. Chapter 23, pp. 331-351. In: Eel Biology, eds. K.Aida, K.Tsukamoto, K.Yamauchi, Springer-Verlag Tokyo, ISBN 4-431-00458-0. Marchelidon J., Le Belle N., Hardy A., Vidal B., Sbaihi M., Burzawa-Gerard E., Schmitz M., Dufour S. (1999) - Etude des variations de paramètres anatomiques et endocrinines chez l'anguille européenne (Anguilla anguilla) femelle, sédentaire et d'avalaison: application à la caractérisation du stade argenté. Bull. Fr. Pêche Piscic. 355, 349-368. Miura, T., Yamauchi, K., Nagahama, Y.,Takahashi, H., 1991a. Induction of spermatogenesis in male Japanese eel, Anguilla japonica, by a single injection of human chorionic gonadotropin. Zool.Sci. 8, 63-73. Miura, T., Yamauchi, K., Takahashi, H., Nagahama, Y., 1991b Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proc. Natl. Acad. Sci. USA 88, 5774-5778. Miura, T., Miura, C.; Yamauchi, K., 2003. Spermatogenesis in the Japanese eel. Chapter 22, pp. 319-331. In: Eel Biology, eds. K.Aida, K.Tsukamoto, K.Yamauchi, Springer-Verlag Tokyo, ISBN 4-431-00458-0. Moberg, G.P., 1985. Influence of stress on reproduction: measure of well-being ? In Animal Stress (ed. G.P. Moberg and M.D. Bethesda) pp. 245-267. Am.Physiol.Soc. Montero M., Le Belle N., King J.A., Millar R.P., Dufour S. (1995) - Differential regulation of the two forms of gonadotropin-releasing hormone (mGnRH and cGnRH-II) by sex steroids in the European female silver eel (Anguilla anguilla). Neuroendocrinology 61, 525-535. Nagae, M., Todo, T., Gen, K., Kato, Y., Young, G., Adachi, S., et al. 1996. Molecular cloning of the cDNAs encoding pituitary glycoprotein hormone a and gonadotropin –II b subunits of the Japanese eel, Anguilla japonica, and increase in their mRNAs during ovarian development induced by injection of chum salmon pituitary homogenate. J.Mol.Endocrinol. 16, 171-181. Nagahama, Y., 1994. Endocrine regulation of gametogenesis in fish. Int. J. Dev. Biol. 38, 217-229. Nilsson, L., Nyman, L., Westin, L., Ornhagen, H., 1981. Simulation of the reproductive migration of European eels (Anguilla anguilla (L.) through manipulation of some environmental factors under hydrostatic compression. Speculations in Science and Technology 4, 475-484.

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Palstra, A.P., Cohen, E.G.H., Niemantsverdriet, P.R.W., van Ginneken, V.J.T., van den Thillart, G.E.E.J.M., 2005. Artificial maturation and reproduction of European silver eel: Development of oocytes during final maturation. Aquaculture 249, 533-547. Pankhurst, N.W., van der Kraak, G., 2000. Evidence that acute stress inhibits ovarian steroidogenesis in rainbow trout in vivo, through the action of cortisol. Gen. Comp. Endocrinol. 117 , 225-237. Pottinger, T.G., Pickering, A.D., 1990. The effect of cortisol administration on hepatic and plasma estradiol-binding capacity in immature female rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 80, 264-273. Pickering, A.D., 1989 Environmental stress and the survival of brown trout, Salmo trutta L. A review. Fresh Water Biology 21, 47-55. Querat, B., Moumni, M., Jusitz, M., Fontaine, Y.A., Counis, R., 1990. Molecular cloning and sequence analysis of cDNA for the putative b subunit of the type-II gonadotropin from the European eel. J.Mol.Endocrinol. 4, 257-264. Robins, C.R., Cohen, D.M., C.H. Robins, 1979. The eels Anguilla and Histio-branchus, photographed on the floor of the deep Atlantic in the Bahamas. Bull.Mar.Sci. 29, 401-405. Rohr, D.H., Lokman, P.M., Davie, P.S., Young, G., 2001. 11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis. Comp. Biochem. Physiol. A 130, 701-714. Romeis, B., 1968. Mikroskopische Technik , R.Oldenbourg Verlag, München, Wien, 757 p Sbaihi,. M Fouchereau-Peron, M., Meunier, F., Elie, P., Mayer, I., Burzawa-Gérard, E., Vidal, B., Dufour, S., 2001. Reproductive biology of the conger eel from the south coast of Brittany, France and comparison with the European eel. J. Fish Biol. 59, 302-318. Schmitz M., Aroua S., Vidal B., Le Belle N., Elie P., Dufour S. (2005). Differential regulation of luteinizing hormone and follicle stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel. Neuroendocrinology 81, 107-119. Sebert, P., Barthelemy, L., 1985. Effects of high hydrostatic pressure per se, 101 atm on eel metabolism. Respiration Physiology 62, 349-357. Simon, B., Sebert, P., Barthelemy, L., 1988. Effects of long-term hydrostatic pressure per se (101 ATA) on eel metabolism. Can. J. Physiol. Pharmacol. 67,1247-1251. Smith, R.J.F., 1985. The Control of Fish Migration (eds. B. Heinrich, W.S. Hoar, K. Johansen, H. Langer, G. Neuweiler and D.J. Randall) , 243 pp. Springer-Verlag, Berlin Heidelberg. Stocco, D.M. 2001. StAR protein and the regulation of steroid hormone biosynthesis. Ann.Rev.Physiol. 63, 193-213. Suetake, H., Okubo, K., Yoshiura, Y., Suzuki, Y., Aida, K., 2002. Differential expression of two gonadotropin (GTH) β subunit genes during ovarian maturation induced by repeated injection of salmon GTH in the Japanese eel Anguilla japonica. Fish.Sci. 68, 290-298. Tesch, F.W., 2003. The eel. Blackwell Science, Oxford (UK). 408pp. Timmers, R.J.M., Lambert, J.G.D., Peute, J., Vullings, H.G.B., van Oordt, P.G.W.J., 1987. Aromatase localization in the brain of the African catfish, Clarias gariepinus (Burchell) by microdissection and biochemical detection. J. Comp. Neurol. 258, 368-377.

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van den Thillart, G., van Ginneken, V., Körner, F., Heijmans, R., van der Linden, R., Gluvers, A., 2004. Spawning migration of the European eel (Anguilla anguilla L.): Endurance swimming of European eel. J. Fish.Biol. 65, 1-7. Vidal, B., Pasqualini, C., Le Belle, N., Holland, M.C.H., Sbaihi, M., Vernier, P., Zohar, Y., Dufour, S. 2004. Dopamine inhibits luteinizing hormone symthesis and release in the juvenile European eel: A neuroendocrine lock for the onset of puberty. Biology of Reproduction 71, 1491-1500. Vøllestad, L.A., 1986. Life-history of the European eel, Anguilla anguilla. A short review. Fauna 39, 117-125. Yoshiura, Y., Suetake, H., Aida, K., 1999. Duality of gonadotropin in a primitive teleost, Japanese eel (Anguilla japonica). Gen.Comp.Endocrinol. 114, 121-131.

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Recommendations for protection of eel populations and suggestions for future research

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Van Leeuwenhoek stelt (in tegenstelling tot de passages hierboven) dat de diertjes aangetroffen in de darmen van paling geen jonge palingen zijn maar wormen Na welke tijd, ik niet alleen verscheijde observatien hebbe gedaan, maar ik hebbe ook veel maal de Visschers, en Ael verkoopers, aangesproken, om uijt haar te verstaan, wat ondervindingen sij mogten hebben ontrent de Voorteelingen vande Alen. Onder dese Menschen vonde ik er twee die oordeelden datse voort teelden, en tot bevestinge bragten sij de volgende redenen voort. “Ontrent inde maant Meij vintmen kleijne roode wormkens inde darmen vande Ael en Paling, en die wormkens (seijden sij) worden Ael en Paling”. Dog alsoo ik veel maal soo danige wormkens, niet alleen uijt de darmen vande Ael en Paling, hadde gehaalt, maar dat ik ook te gelijk die wormkens hadde ontledigt, ende gesien, dat die wormkens selfs, een groote menigte van wormkens in haar lighaam hadden, en dat over sulks die wormkens selfs bij inlijvinge voorteelden, soo heb ik soo danig seggen mede moeten verwerpen, te meer om dat ik onder vond, dat dese wormkens haar seer vast hegten, inde darmen vande Ael en Paling, om niet met de Chijl, die uijt de darmen als excrementen getsooten werden mede souden uijt gedreven werden. In somma, men moet dese kleijne wormen, voor een ongediert der Darmen, vande Ael en Paling aan nemen, gelijk wij doen, de wormen die inde Darmen van verscheijde dieren gevonden werden. (Antoni van Leeuwenhoek, Brief No. 123 [75], 16 september 1692). Chapter 14

Recommendations for protection of the eel populations and future research

1) Eel fisheries Human impact on downstream migrating silver eel in European inland waters mainly consists of withdrawal of eel by commercial fisheries. Therefore eel fisheries, is still the major source for diminishing the eel population. Bruijs et al. (2003) estimated that eels in the river Meuse have a chance of at least 30% and probably about 40% to reach the North Sea. In this river, the eel mortality due to commercial eel fisheries was 22,2% while mortality due to two hydropower stations was 15,8% (Bruijs et al. 2003). Importance of eel fisheries can be concluded from the observation that for Europe at least 25,000 people generate at least part of their income from eel fisheries and eel aquaculture (Dekker 1998). Most biologists agree that eel fisheries on mature migrating silver eels should be halted as well as fisheries on glass eel should be strictly regulated. These restrictive measurements for eel fisheries should be taken for Europe in an international context and if we also consider other eel species than the European eel (Anguilla anguilla L.) should be applied on a worldwide scale

2) Habitat degradation and losses due to hydropower stations For the European eel (Anguilla anguilla) habitat degradation often brought about by human activity, can result in fish mortalities and may result in prevention of adult spawners to reach the ocean. An example for habitat degradation for the adult spawners is the result of barriers in the migration routes such as dams, sluices, gauging structures (Legault 1990). Also passing of turbines of hydropower facilities by eel may result in an extra mortality of eel (Bruijs et al. 2003). As a consequence these human activities might have detrimental effects on the population level of the European eel (Bruijs et al. 2003). Development of early warning systems in order to protect a substantial part of the downstream migrating silver eels is a prerequisite for management of the eel population. In this respect, the development and application of the Nedap Trail system®, a telemetry transponder system, which consist of ‘detection lines’ across the bottom of the river, is an example of an appropriate ‘early warning system’. With this system waves of tagged downstream migrating silver eel can be observed. Consequently turbine management can take into account these migration patterns by temporarily closing down the turbines (Bruijs et al. 2003).

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Another very promising monitoring system is the Migromat® system, which very accurately registers the pre-migratory restlessness of eels. With this system during short experimental periods of several weeks in 2002 the eel mortality due to passing the hydropower stations could be reduced with approximately 70% (Bruijs 2003).

3) Shortage of fat stores due to insufficient food supply in the inland waters Direct proof that European eels are able to swim the long distances between Europe and the Sargasso Sea was until now missing. Tucker (1959) had severe doubts whether the European eel would be able to swim on its energy reserve stores across the ocean and suggested that all European eels are the offspring of the American eel. In our work we demonstrate, measuring the energy costs of migration via two independent methods, that eel swim 4 to 6 times more efficiently than many other fish species, even across swimming styles. The Cost of Transportation (COT) for eel was 0.68 kJ.kg-1.km-1 while the COT for trout was 2.73 kJ.kg- 1.km-1. The estimated fat use for an adult eel to cross the Atlantic (6,000-km) would be 29% of its fat stores corresponding to 58 g fat/kg eel while this would be for salmon 300 g/kg. Eels have a fat content of 10-28% with a mean of 20%, which is obviously the predominant energy store. 60% of the total fat reserve of silver eels is required for swimming. Animals with less than 13% fat would not be able to swim 6000-km. Based on these observations we can conclude that due to the low energy costs of migration the energy reserves possibly may not become a limitation to reach the Sargasso Sea. In practice in only one study low energy fat stores were reported in eels prior to migration (Svedäng & Wickström 1997). Therefore we consider limited food stores not as a major threat for eel populations.

4) Contamination of the inland waters with PCB’s PCBs are persistent pollutants, which will remain in the aquatic environment for the coming several decades despite the ban on its usage in Europe. PCBs accumulate in the fat stores of the eels. Our study showed that weight loss in PCB loaded eels was 1.5 times higher in both swim- and rest- groups in comparison to respectively Control swim- and rest-groups. Oxygen consumption of PCB loaded animals was significantly lower (P≤0.0001) after migrating 750 km in comparison to the control swim group. This can possibly be ascribed to a suppressed protein synthesis. Also a hypoglycemia was observed in both PCB-swim and rest group. This can possibly be ascribed to an altered carbohydrate metabolism of PCB-exposed animals.

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In addition, our studies indicate that PCBs have deleterious effects on the fertility by impairing egg quality and subsequent embryonic development (Palstra et al. 2005). Furthermore, long-term swimming will increase the levels of PCBs due to specific usage of the fat stores, which will increase the PCB level in the blood and result in even lower egg quality. We recommend that PCB contamination should be monitored in all major hydro- systems and that areas with low PCB levels should be protected. Actions to reduce the levels to a minimum should be undertaken to improve the water quality and protect our aquatic ecosystem.

5) Virus infections Human activity including aquaculture practices has greatly enhanced global transport of fish species including pathogens from transfer of infectious stocks. Within the last few decades, aquaculture has become an important aspect of our society. Its global production has more than doubled in weight and value between 1986 and 1996 and over one quarter of human fish-consumption is produced in aquaculture (Naylor et al. 2000). With the growing amount of in aquaculture produced products, transfer of diseases by transport of stock and food supplies has increased. Blanc (1997) points out that nearly one hundred pathogens have been introduced in European hydrosystems since the introduction of aquaculture. In the in this thesis presented work many virus-infections were found in eel populations in the Netherlands, which is a threat for the whole eel-population as the Netherlands is one of the leading eel-trading countries (Heinsbroek & Kamstra 1995). Widespread infection of the eel- population with the EVEX virus may result from unlimited intercontinental transport. An alarming situation is that EVEX was found in our study in several countries worldwide. For the European eel (Anguilla anguilla) in the Netherlands, Italy and Morocco but even in another eel species than European eel: the New Zealand eel (Anguilla dieffenbachi). In this respect it is worrying that also Herpesvirus anguillae is isolated and identified in eel populations all over the world. In cultured eel in Taiwan (Ueno et al. 1992, 1996 Chang et al. 2002), in cultured eels in the Netherlands (our results, van Nieuwstadt et al. 2001, Davidse et al. 1999) but also in our study in adult European eel from Lake Grevelingen. Recommendations to cope with the virus problem are twofold: a) Global instructions and sanitary standards for transportation of aquatic animals set up by international organizations should be introduced, in order to avoid further risks for animal health. b) New diagnostic tools are needed for identification, isolation and characterization of possible causative agents like species-specific DNA probes (Harvell et al. 1999).

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6) Reproduction, stripping experiments Regrettable, our 'stripping' experiments did not result in the production of vital larvae. Therefore the following recommendations can be made for follow up research. First, a method has to be found to administer carp pituitary at a regular basis during a period of 3-4 months. In the performed study we gave weekly a bolus injection at a level of one million times above the physiological dosage. The analysis of the kinetics of GTH (LH like) clearance indicated a curve with two components (Dufour 1985). The first component (phase of rapid clearance) was indicative for an initial half-life of 32-45 minutes. The second phase (up to about 24 hours), was indicative for a half-life of about 4-8 hours. The analysis of the whole curve indicated a metabolic clearance of 12-20 ml/hour/kg. The study was conducted on catheterized female silver eels, in freshwater at 18 ± 2 °C, using cold or radioactive pure carp GTH (Dufour 1985). From the half-life time of approximately 5 hours (Dufour 1985) we can conclude that this pharmacological dosage leads to enormous differences in the concentrations of cPS. This is confirmed by the pharmacokinetic study of Sato et al. (1995) where there was a total clearance of administered gonadotropines after 24 hours. An injection interval of one until two times per week is therefore insufficient to maintain the appropriate gonadotropine level. A) Possibly peristaltic infusion pumps can be a solution for a more regularly administration protocol of the cPS. B) A second improvement can be concerning the issue of ovulation. If injection of the ovulation hormone 17α-20β dihydroxyprogesteron directly in the ovary (Ohta et al. 1996, 1997, Lokman & Young 1995, 2000) in future studies doesn’t lead to the production of larvae, it has to be reconsidered to work at the level of the hypothalamus-pituitary by administration of gonadotropine-releasing hormone (GnRH) and dopamine blockers. These techniques have until now not been applied for eel but have in Europe been developed for African catfish (De Leeuw et al. 1985, Richter et al. 1982, 1985, 1987). With respect to the neuroendocrine control of the eel gonadotropic function, recently the actual involvement of Dopamine in the control of puberty in the European eel has been clarified and assessed (Vidal et al. 2004, Dufour et al. 2005). Working at the level of the neuroendocrine control of the eel gonadotropic function is a more natural situation because the animal can determine itself the moment of ovulation which is a much more natural situation. Ultimately this will be more beneficial for the quality of the eel larvae (Peter and Yu 1997).

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C) A third recommendation concerns the behavioural aspect, which is described in annex 2. In the Sargasso Sea, sexual activity is probably limited to a restricted period. Therefore the location of suitable mates, courtship and spawning must take place quite quickly. Probably spawning is thus synchronised throughout the population. Information on spawning behaviour of European eel has to our knowledge never been published before. Natural spawning may be stimulated due to the action of pheromones (Colombo et al. 1982), which may have a positive effect on hatching and fertilisation rates. Therefore spawning in groups instead of stripping of animals may be a more natural situation.

7) Reproduction, natural trigger The life-cycle of the European eel still holds many mysteries. The catadromous European eel migrates 6,000-km from its freshwater habitats to the Sargasso Sea where it spawns, following which the adults die. To date, a full understanding of the mechanisms controlling sexual maturation in the eel is still lacking. In the migratory silver stage of the European eel it has been demonstrated that a prepubertal blockage occurs in the hypothalamus-pituitary- gonad (HPG) axis, resulting in the inhibition of sexual maturation. It has been proposed that the unique environmental factors experienced by the eel during its prolonged migration may in some way influence reproductive development (figure 1).

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Figure 1: Schematic overview of the hypothalamus-pituitary-gonad (HPG) axis. Its blockage and its stimulation by environmental triggers (after: Dufour 1994)

In our studies we found that female eels, allowed to swim constantly for 5,500-km over a 5-6 month period, showed more advanced gonadal maturation compared to non-swimming controls. The eels allowed to swim showed elevated plasma levels of gonadotropin and 17β- estradiol, and oocytes at a more advanced stage of development. However these animals came from a hatchery, were in the yellow stage, and only a few years old. Therefore we recommend to perform in future work similar migration studies with older animals.

8) Molecular Work In order to find the natural trigger for maturation and in order to elucidate which genes are ‘switched on’ after an experimental protocol like e.g. swimming we can make use of another fish species, the zebrafish (Danio rerio). For this fish species the sequencing of its genome is almost completed (http://www.ensembl.org/Danio_rerio/). Using the list of candidate eel (Anguilla sp.) genes as query subjects, a BLAST (Basic Local Alignment

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Search Tool) comparison can be performed to search against the zebrafish genome (Danio rerio). The zebrafish species has become an established model organism to study vertebrate development, since it facilitates a improved way to perform forward genetic screens (Driever et al. 1994, Talbot & Hopkins 2000, Peterson et al. 2000, Haffter et al. 1996, Driever et al. 1996, Gollig et al. 2002, Mullins 2002, Amsterdam et al. 1999). Furthermore, the genome of this teleost contains a complete vertebrate gene set whose products establish fundamental developmental processes (Driever et al. 1994, Ekker & Larson 2001). Zebrafish have short generation times as well as high external fertilisation rate and its embryos are transparent (i.e. optically clear) which also develop externally very quickly (Chen & Ekker 2004, Ekker 2000, Ekker & Larson 2001). Therefore zebrafish is an excellent organism in developmental genetic screens. The strategy of performing such a BLAST comparison analysis against the genome of zebrafish has two objectives: a) To determine the percentage of identical amino acids in this BLAST search procedure, meaning that the query eel amino acid sequence contains several identical amino acids when compared to (particular) gene-chunk transcripts across the zebrafish genome. b) The chunks of the zebrafish genome in which there is a significant homology with the eel query (i.e. 70% or greater positive amino acid homology), should be checked against a database containing several zebrafish micro-array layouts in order to identify these several genome chunks. The latter strategy is important because the percentage of positive amino acid homology between the eel protein transcripts and zebrafish genome chunks gives an idea of how many amino acids are not identical, but have similar physical properties. We were able to identify several zebrafish genes using version Zv3 of zebrafish genome database (http://www.ensembl.org/Danio_rerio/), which obviously have homology with eel (Anguilla sp.) counterparts (see table 1 and table 2).

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Tabel 1: Comparison of the genes of eel (Anguilla sp.) with zebrafish (Danio rerio) based on DNA/mRNA sequence. BLAST search data (table of means) Anguilla DNA/mRNA seq. vs. Genomic contigs Danio rerio (zebrafish) Anguilla Name Protein aa-sequence homology with Zf genome species (derived from mRNA/DNA) chunks % id ± STDEV (%) % pos ± STDEV (%) (mean) id (mean) pos thyrotropin beta subunit No Hits No Hits No Hits No Hits (variant A14, 3'-UTR) mRNA thyrotropin beta subunit 73.63 2.43 73 3 (variant P22, 3'-UTR) mRNA TSHB gene (variant 2S) ang. 62.23 2.26 62 2 DNA TSHB gene (variant 4S) 60.50 2.55 60 3 DNA TSHB gene (variant 9L) 61.11 2.67 61 3 DNA GnRH receptor jap. intron within 59.50 2.46 59 2 Intracellular Domain 3 mRNA GnRH receptor jap. intron within 58.25 2.41 58 2 Transmembrane Domain 4 mRNA

From table 2 it can be concluded that the mean % of identical amino acids for all examined proteins corresponds to 61.08 % ± 0.87 while the number of positive amino acid homology has a similar number, 61.0 % ±1. This is a rather low number, is disappointing and reflects the fact that eels are not much related with zebrafish. This can also be concluded from figure 1, which gives a tree of commonly accepted relationships between inter alia the different Teleosts species (Nelson 1994, Pichon & Ghysen 2004).

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BLAST search data (table of means) A nguilla Protein seq. vs. Genomic contigs Danio rerio (zebrafish) Anguilla Name Protein aa-sequence homology with Zf genome sp ecies (derived from mRNA/DNA) chunks % id ± STDEV (%) % pos ± STDEV (%) (mean) id (mean) p os Gonadotropin type II 50.92 9.12 68 10 beta subunit ang. Gonadotropin type II 50.92 9.12 68 10 beta subunit p recursor Gonadotropin type II rostr. 50.92 9.12 68 10 beta subunit p recursor Gonadotropin type II 50.92 9.12 68 10 beta subunit p recursor jap. Gonadotropin type I 48.70 8.16 62 6 beta subunit Growth ang. 51.67 12.06 68 6 hormone (GH) Growth jap. 52.22 12.22 69 6 hormone (GH) FSH beta 49.16 8.19 62 6 subunit ang. FSH beta 50.76 10.58 64 9 subunit (GTH I) Somatolactin 49.98 6.68 73 5 p recursor ang. Glycoprotein 56.80 5.20 71 4 alp ha subunit p recursor Prolactin ang. 63.25 11.98 79 12 p recursor Prolactin jap. 65.45 8.72 84 8 p recursor ang. Pit-1 protein 62.24 19.22 74 15 prepro-ghrelin 49.97 15.37 86 12 jap. Proopiomelanocortin 47.53 10.04 61 12 rostr. Proopiomelanocortin I 46.04 8.35 57 10

11beta HSD 53.92 16.60 67 15 prepro-cGnRH-II 58.96 11.47 74 13 (DNA and mRNA) prepro-mGnRH 41.41 10.60 61 11 (DNA and mRNA) activin B 44.43 16.38 61 13 beta-actin 80.65 16.05 87 11 Spermatogonial stem-cell renewal factor 58.08 17.09 73 16 (esrs34e10-5 mRNA) jap. spermatogenesis preventing substance 36.12 12.28 53 14 (esrs21c19 mRNA) Steroidogenic acute regulatory protein (StAR) 55.42 19.81 74 15 (mRNA) lip gene for triglyceride 39.57 12.85 54 9 lip ase androgen receptor 58.61 15.41 73 14 alp ha androgen receptor 61.59 16.65 75 15 beta Tabel 2: Comparison of the genes of eel (Anguilla sp.)with zebrafish (Danio rerio) based on protein sequence.

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However, despite the low number of homology between the genes of zebrafish (Danio rerio) and eel species (Anguilla sp) it is worth to do the following three experiments in order to further investigate which eel genes influence the sexual maturation of the several eel species. Following the first two relatively necessary steps using zebrafish as a model and starting point, several strategies can be followed in future studies to further investigate which eel genes influence the sexual maturation of the several eel species. Firstly, a new comparison analysis should be performed using a more recent version of the zebrafish genome database, which may give more accurate results and even more eel candidate genes might identified for further molecular testing, when a new BLAST search is performed using the latest available zebrafish genome database (Assembly Zv5 (released may 27th 2005); database v36.5c, dated 5th December 2005; http://www.ensembl.org/Danio_rerio/). In this matter even a greater percentage of identical amino acid sequences next to a possible increase of positive amino acid sequences might be found, as it is of a great importance of further molecular testing of eel gene constructs. The next step in this approach would then be to use the newly identified zebrafish genome to identify functional zebrafish proteins in a database containing several zebrafish micro-array layouts in order to identify these several genome chunks (i.e. annotation of known zebrafish protein sequences to the found genome chunks in BLAST search). Secondly, an useful experiment would be to clone several eel candidate gene constructs originating from interesting tissues (e.g. ovary tissue, pituitary cells) in several specific according zebrafish gene constructs (coding for the tissue cell types such as those from the head region and/or pituitary cells) in conjunction with a useful reporter (like GFP, luciferase, etc.) as described in a similar experiment (Udvadia & Linney 2003) and transfer these resulting transgenic zebrafish cells back to a zebrafish embryo. In this way two lines of eel cells could be used, since one line of cells would originate from eels which have been monitored during a swim tunnel experiment, and another line of cells would come from fishes which have not taken part in such a swim tunnel experiment. Then by looking at the transgenic zebrafish (by screening fluorescence levels in zebrafish embryos), one could tell if the transgenic “eel-zebrafish construct” is showing a difference in expression when two different eel lines have been used. In other words, there is a possibility that certain eel genes are up- or downregulated as a result of the swim tunnel protocol. Using this approach the exact eel gene can subsequently be identified with no great effort needed.

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Thirdly, the use of micro-arrays specifically designed for eel can also serve as an adequate approach to the genetic background of the eel sexual maturation process. The DNA micro-array is an assay that can be used to measure the expression level for many genes (up to thousands or more) simultaneously in a collection of cells. In this manner this kind of assay can give some understanding of functionality of certain cell types on a genome wide scale. Different cell lines from male and female eels which have undertaken a swim-tunnel experiment and as a control group those which have not (e.g. pituitary cells, and others) can be assigned as constituents of both the “first step" (Churchill 2002) (the fishes as biological/experimental units divided randomly into swimming or no-swimming treatment groups) as well as the “second step” (Churchill 2002) (RNA samples collected of each group and random assignment of dye labels to the RNA sample groups) in the design of a two-colour eel micro-array study for example. The "third step" in this particular eel micro-array design (Churchill 2002) would then consist of the genes from the candidate list (when known from which eel cell tissue type they originate from) to be spotted on a series of pre-fabricated micro arrays to see which are been up- or down-regulated during a swim- tunnel and which have not. In this way the exact genes could be determined. The potential of the two-colour micro-array system is therefore fully exploited as it provides the ability to make direct comparisons between two samples on the same micro-array slide (Churchill 2002).

9) Sender satellite techniques: animal tracking studies The theory that all European eel migrate to the Sargasso Sea for reproduction and compromise a single randomly mating population (the panmixia theory) needs definitely to be confirmed or rejected. Also, if the Sargasso Sea is the spawning area of the European eel, it needs to be elucidated what makes this area so unique. Satellite tracking studies with the Argos satellite system, a telemetry system specially developed for animal tracking studies, are very promising (Taillade 1992). The most interesting potential contribution of telemetry tracking from silver eels is knowledge about routes, rates and depths of travel. Miniaturization of the telemetry equipment may enable us in future studies to track small eel species (between 1-2 kg) like the European, American and Japanese eel.

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LITERATURE

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Driever W.; Solnica-Krezel L.; Schier A.F.; Neuhauss S.C.F.; Malicki J.; Stemple D.L.; Stainier D.Y.R.; Zwartkruis F.; Abdelilah S.; Rangini Z.; Belak J.; Boggs C. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123: 37-46.

Dufour, S. (1985). La fonction gonadotrope de l'anguille européenne, Anguilla anguilla L., au stade argenté (au moment du départ pour la migration de reproduction): les mécanismes de son blocage et sa stimulation expérimentale. (1985) Thèse de Doctorat

Dufour, S. (1994). Neuroendocrinologie de la réproduction de l'anguille: de la recherche fondamentale aux problèmes appliqués. Bulletin Français de la pêche et de la pisciculture 335: 187-211.

Dufour, S.; Weltzien, F-A.; Sebert, M-E.; Le Belle, N.; Vidal, B.; Vernier, P.; Pasqualini, C. (2005). Dopaminergic inhibition of reproduction in Teleost Fishes: Ecophysiological and Evolutionary implications. Ann.N.Y.Acad.Sci. 1040: 9-21.

Ekker S.C. (2000). Morphants: a new systematic vertebrate functional genomics approach. Yeast 17: 302-306.

Golling G.; Amsterdam A.; Sun Z.; Antonelli M.; Maldonado E.; Chen W.; Burgess S.; Haldi M.; Artzt K.; Farrington S.; Lin S.-Y.; Nissen R.M.; Hopkins N. (2002). Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat. Genet. 31: 135-140.

Haffter P.; Granato M.; Brand M.; Mullins M.C.; Hammerschmidt M.; Kane D.A.; Odenthal J.; van Eeden F.J.M.; Jiang Y.-J.; Heisenberg C.-P.; Kelsh R.N.; Furutani-Seiki M.; Vogelsang E.; Beuchle D.; Schach U.; Fabian C.; Nüsslein-Volhard C. (1996). The identification of genes with unique and essential functions in development of zebrafish. Development 123: 1-36

Heinsbroek, L.T.N.; Kamstra, A. (1995). The River Eels, Chapter 6: In: Production of Aquatic Animals (eds. C.E. Nash and A.J. Novotny) pp. 109-131, Elsevier, Amsterdam.

Legault, A.(1990). Estuary dams management and eel migration. Int.Rev.Ges.Hydrobio 75:819-825.

Lokman, P.M. & Young, G. (1995). In vitro biosynthesis of oestradiol-17β and 17α,20β dihydroxy-4-pregnen-3-one by vitellogenic ovarian follicles from migrating New Zealand longfinned eels (Anguilla dieffenbachii). Aquaculture 135: 17-26.

Lokman, P.M. & Young, G.( 2000). Induced spawning and early ontogeny of New Zealand freshwater eels (Anguilla spp.) . New Zealand Journal of Marine and Freshwater Research 34: 135-145.

Mullins M.C. (2002). Building-blocks of embryogenesis. Nat. Genet. Suppl. 31: 125- 126.

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Naylor, R.L.; Goldburg, R.J.; Primavera, J.H.; Kautsky, N.; Beveridge, M.C.C.; Clay, J.; Folke, C.; Lubchenco, J.; Mooney, H.; Troell, M.(2000). Effect of aquaculture on world fish supplies. Nature 405: 1017-1024

Nelson, J.S. (1994). Fishes of the World. 3d Ed. John Wiley & Sons, New York.

Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., & Hirose, K. (1996). Changes in fertilization and hatching rates with time after ovulation induced by 17α, 20β-dihydroxy-4- pregnen-3-one in the Japanese eel, Anguilla japonica. Aquaculture 139: 291-301.

Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N. & Hirose, K. (1997). Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica. Fish Physiology and Biochemistry 17: 163-169.

Palstra, A.P., van Ginneken, V.J.T., Murk, A.J. and van den Thillart G.E.E.J.M. (2005). Are dioxin-like contaminants responsible for the eel (Anguilla anguilla) drama ?. Naturwissenschaften submitted.

Peter, R.E. & Yu, K.L. (1997). Neuroendocrine regulation of ovulation in fishes: Basic and applied aspects. Reviews in Fish Biology and Fisheries 7: 173-197.

Peterson R.T.; Link B.A.; Dowling J.E.; Schreiber S.L. (2000). Small molecule developmental screens reveal the logic and timing of vertebrate development. Proc. Natl. Acad. Sci. USA 97: 12965-12969

Pichon, F. & Ghysen, A. (2004). Evolution of posterior line development in fish and amphibians. Evolution & Development 6: 187-193.

Richter, C.J.J. & van den Hurk, R. (1982). Effect of 11-desoxycorticone-acetate and carp-pituitary suspension on follicle maturation in the ovaries of the African catfish, Clarias lazera (C&V). Aquaculture 29: 53-66.

Richter, C.J.J., Eding, E.H. & Roem, A.J. (1985). 17α-hydroxy-progesterone induced breeding of the African catfish, Clarias gariepinus (Burchell), without priming with gonadotropin. Aquaculture 44: 285-293.

Richter, C.J.J., Eding, E.H, Goos, H.J.Th, De Leeuw, R. Scott, A.P. & Van Oordt, P.G.W.J. (1987). The effect of pimozide-LHRHa and 17α hydroxy-progesterone on plasma steroid levels and ovulation in the African catfish, Clarias gariepinus. Aquaculture 63: 1571-68.

Sato, N., Kawazoe, I. , Shiinna, Y., Purukawa, K., Suzuki, Y., & Aida, K. (1995). A novel method of hormone administration for inducing gonadal maturation in fish. Aquaculture 135: 51-58.

Svedäng, H.; Wickström, H. (1997). Low fat contents in female silver eels: indications of insufficient energetic stores for migration and gonadal development. J.Fish Biol. 50: 475-486 .

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Taillade, M.(1992). Trends in satellite based animal tracking. Biotelemetry X11 (Service Argos).

Talbot W.S.; Hopkins N. (2000). Zebrafish mutations and functional analysis of the vertebrate genome. Genes Dev. 14: 755-762

Tucker, D.W. (1959). A new solution to the Atlantic eel problem. Nature 183: 495-501. Van Nieuwstadt, A.P.; Dijkstra, S.G.; Haenen, O.L.M. (2001). Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla. Diseases of Aquatic Organisms 45: 103-107.

Udvadia A.J.; Linney E. (2003). Windows into development: historic, current and future perspectives on transgenic zebrafish. Dev. Biol. 256: 1-17

Ueno, Y.; Kitao, T.; Chen, S-N., Aoki, T.; Kou, G-H. (1992). Characterization of a Herpes- like Virus isolated from cultured Japanese eels in Taiwan. Gyohyo Kenkyu 27: 7-17.

Ueno, Y.; Shi, J.-W.; Yoshida, T.; Kitao, T.; Sakai, M.; Chen, S.-N.; Kou, G.H.(1996). Biological and serological comparisons of eel herpesvirus in Formosa (EHVF) and herpevirus anguillae (HVA). J.Appl.Ichthyol. 12: 49-51.

Vidal, B.; Pasqualini, C.; Le Belle, N.; Holland, M.C.H.; Sbaihi, M.; Vernier, P.; Zohar, Y.; Dufour, S. (2004). Dopamine inhibits luteinizing hormone synthesis and release in the juvenile European eel: A neuroendocrine lock for the onset of puberty. Biology of Reproduction 71: 1491-1500.

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The European eel (Anguilla anguilla, Linnaeus), its lifecycle, evolution and reproduction: a literature review.

Vincent J.T. van Ginneken1 and Gregory E. Maes2.

1 Institute Biology Leiden, Integrative Zoology,van der Klaauw Laboratorium, POB 9511, 2300RA,Leiden, The Netherlands, [email protected], +31(0)71-5274900 2 Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Ch. Deberiotstraat 32, B-3000, Leuven, Belgium

Key words: Anguilla, migration, Sargasso Sea, molecular studies, spawning behaviour, satellite

Running Title: European eel lifecycle, evolution and reproduction

Reviews in Fish and Fisheries: in press

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Fixatie van een paling om de bloedsomloop te bestuderen Op een ander tijd heb ik een Ael bij na geheel int water gestelt, en het hooft in sulken naeuwte gebragt of geklemt, dat de Ael sijn kaken gans niet bewegen konde, en in die benautheijt sijn mond bleef open houden, ende als doen het bloet observerende, sag ik (na verloop van eenigen tijd) dat verscheijde malen het bloet inde Aderen op het eijnde vande staart, veel maal voor een oogenblik tijds, gans bleef stil staan, en dat daar op dan een kleijne voortstootinge tot twee à. drie maal agter den anderen volgde, waar na dan het bloet voor een korten tijd, sijn voorgaande cours nam: en diergelijke voorval geschiede in dese Ael verscheijde maal in weijnig tijds. (Antoni van Leeuwenhoek, Brief No. 113 [66], 12 januari 1689).

Annex 1

The European eel (Anguilla anguilla, Linnaeus), its lifecycle, evolution and reproduction: a literature review.

Abstract: The European eel (Anguilla anguilla Linnaeus 1758) is a species typical for waters of Western Europe. Thanks to early expeditions on the Atlantic Ocean by the Danish biologist Johannes Schmidt who found small (< 10 mm) leptocephali larvae in the Sargasso Sea about 100 years ago, we have now a strong indication where the spawning site for this species is located. The American eel (Anguilla rostrata, LeSueur) also spawns in the Sargasso Sea. The spawning time and location of both species have been supported and refined in recent analyses of the available historical data. Subsequent ichthyoplankton surveys conducted by McCleave (USA) and Tesch (Germany) in the 1980s indicated an increase in the number of leptocephali < 10 mm , confirming and refining the Sargasso Sea theory of Johannes Schmidt. Distinctions between the European and American eel are possible based on as well morphological characteristics (number of vertebrae) as molecular markers (allozymes, mitochondrial DNA and anonymous genomic-DNA. Although recognised as two distinct species, it remains unclear which mechanisms play a role in species separation during larval drift, and what orientation mechanism eels use during migration in the open sea. The current status of knowledge on these issues will be presented. The hypothesis that all European eel migrate to the Sargasso Sea for reproduction and comprise a single randomly mating population, the so called panmixia theory, was until recently broadly accepted. However, based on field observations, morphological parameters and molecular studies there are some indications that Schmidt's claim of complete homogeneity of the European eel population and a unique spawning location may be an overstatement. Recent molecular work on European eel indicated a genetic mosaic consisting of several isolated groups, leading to a rejection of the panmixia theory. Nevertheless, the latest extensive genetic survey indicated that the geographical component of genetic structure lacked temporal stability, emphasising the need for temporal replication in the study of highly vagile marine species. Induced spawning of hormone treated eels in the aquarium was collective and simultaneous. In this work for the first time group spawning behavior has ever been observed and recorded in eels.

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Studies in swim-tunnels indicate that eels can swim four to six times more efficiently than non-anguilliform fish such as trout. After a laboratory swim trial of eels over 5,500-km, the body composition did not change and fat, protein and carbohydrate were used in the same proportion. This study demonstrated for the first time that European eel are physiologically able of reaching the Sargasso Sea without feeding. Based on catches of newly hatched larvae, temperature preference tests and telemetry tracking of mature hormone treated animals, it can be hypothesised that spawning in the Sargasso Sea is collective and simultaneous, while presumably taking place in the upper 200 meters of the ocean. Successful satellite tracking of longfin female eels in New Zealand has been performed to monitor migration pathways. Implementation of this new technology is possible in this species because it is three times larger than the European eel. In the future, miniaturization of tagging technology may allow European eels to be tracked in time by satellite. The most interesting potential contribution of telemetry tracking of silver eels is additional knowledge about migration routes, rates, and depths. In combination with catches of larvae in the Sargasso Sea, it may elucidate the precise spawning locations of different eel species or groups. Only then, we will be able to define sustainable management issues by integrating this novel knowledge into spawners escapement and juvenile fishing quota.

Introduction

Although a large amount of scientific literature has been produced on freshwater eels (Anguilla sp.; see e.g. references of this review), major questions still have to be resolved mainly on the topic of spawning grounds and reproduction. Already around 350 BC Aristotle's wrote in his 'Historia Animalium': "the eels come from what we call the entrails of the earth. These are found in places where there is much rotting matter, such as in the sea, where seaweeds accumulate, and in the rivers, at the water's edge, for there, as the sun's heat develops, it induces putrefaction." (Bertin 1956). Until the early 20th century, one could reasonably speak of the mysterious life of the eel. Thanks to the early marine expeditions of the Danish biologist Johannes Schmidt (see figure 2 for sampling stations for larvae) the central mystery of its breeding location has been elucidated (Schmidt 1922, 1923, 1925, 1935). Schmidt based his conclusion regarding the spawning site of the European eel in the Sargasso Sea (figure 1) on larvae (Lepocephali) distributions (see section “The location of the spawning areas”).

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Despite the intensive research on eels following the work of Schmidt (1923, 1925, 1935), there are many uncertainties, and there is still a lack of knowledge on many aspects of the life cycle of the European eel. This is best summarised in the book of Harden Jones (1968): "No adult eels have ever been caught in the open Atlantic nor eggs definitely identified in the wild. Migration routes and spawning conditions for adults are unknown or conjectural, as are many details of the development, feeding and growth of larvae. Mechanisms for species separation (note: separation between the American eel and the European eel) during larvae migration are speculative, and details of larval migration or drift are uncertain". In this review we will present the progress in knowledge and new insights about the eel life cycle following the initial work of Schmidt at the beginning of the previous century. This new information is based on the application of new techniques and methodologies such as refined and improved catching techniques for ichthyoplankton surveys, new molecular DNA analyses, telemetry-tracking studies, endocrinological surveys in field studies, energy balance studies in large swim-tunnels, and behavioural studies of hormone treated animals.

Figure 1: Distribution patterns of eel larvae with the size of the larvae in mm (source: Schmidt 1923).

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Figure 2: Principal Danish collection stations of eel larvae, 1903-1922 (After: Schmidt 1925). Closed circles indicate stations by research ships and open circles those by other ships (source: Vladykov 1964).

1) Eel life cycle and fisheries

The life-history of the European eel (Anguilla anguilla L.) depends strongly on oceanic conditions; maturation, migration, spawning, larval transport and recruitment dynamics are completed in the open ocean (Tesch, 2003). Partially mature adults leave the continental rivers at different times, strongly dependent on lunar phase and atmospheric conditions (Desaunay & Guérault, 1997; Okamura et al., 2000; Tesch, 2003), swim southward using the Canary and North-Equatorial currents and arrive six to seven months later at the Sargasso Sea to spawn and then die. The leptocephali larvae are transported along the Gulf Stream and North-Atlantic Drift for a journey of eight to nine months back to the eastern Atlantic coast

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(Lecomte-Finiger, 1994; Arai et al., 2000), where they metamorphose to glass eels, ascent rivers and grow till partial maturity, six to ten years later (Tesch, 2003).

A total of 25,000 tons of eels are consumed in Europe annually (Usui 1991). Eel fisheries in Europe cover an area of 90,000 km2 with approximately 25,000 people generating income from eel fisheries and aquaculture (Dekker 1998, 2003a, 2004). On a worldwide scale eel (fisheries and fish culture) was estimated to produce between 100,000 to 110,000 tons in 1987, which corresponds to approximately 2-2.2 billion Euros per year (Heinsbroek 1991). Eel populations have been declining worldwide over the last decade (Stone 2003). European eel (Anguilla anguilla) numbers have dropped as much as 99% since the early eighties of the previous century, while Japanese eel (Anguilla japonica) dropped as much as 99% since the early seventies of the previous century (Dekker, 2003b). North-American eels are suffering steep drop-offs as well (figure 3A). Also the trends in glass eel recruitment to the European continent show steep declines from the eighties of the previous century (figure 3B). The exact cause for this phenomenon is unknown, but possible causes include: a) contamination with toxic PCBs, which are released from fat stores during their long-distance migration and interfere with reproduction (Castonguay et al. 1994); (b) infection with the swimbladder parasite Anguillicola crassus (Haenen 1995); (c) viruses (van Ginneken et al. 2004, 2005a), (d) oceanographic/climatic changes (Knights 2003); (e) diminished fat stores due to insufficient food supplies in the inland waters (Svedäng and Wickstrom 1997); (f) blockage of migration routes by power stations and plants (Castonguay et al. 1994); and (g) over-fishing (Castonguay et al. 1994, Dekker 2003a, 2004).

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Figure 3A: Time trends in juvenile abundance of the major eel stocks of the world. For Anguilla anguilla, the average trend of the four longest data series is shown for A.rostrata, data represent recruitment to Lake Ontario; for A.japonica, data represents landings of glass eel in Japan (Source: Dekker 2003b, 2004).

Figure 3B: Trends in glass eel recruitment to the continent. Individual data series are given in grey; common trend (geometric mean of the three longest data series in black. Data from ICES (2004) and Hagström and Wickström (1990) (source: Dekker 2004).

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2) The location of the spawning areas Information about the exact location of the spawning grounds can be acquired based on catches of larvae eels in relation to size and age. Johannes Schmidt gathered records of over 10,000 European eel larvae and about 2,400 American eel larvae over a period of 25 years. Schmidt based his conclusions about the oceanic life history of eels on the spatio-temporal distribution of larvae of different sizes. He never captured adult eels in the open ocean en route to or in the Sargasso Sea. Furthermore, eel eggs still have not been identified in plankton samples from the Sargasso. Schmidt reached the conclusion that the European eel only spawns in the Sargasso Sea in the south-western portion of the North Atlantic Ocean from the distribution of the smallest larvae (Schmidt 1923). This until recently well-accepted conclusion about a single spawning area in the Sargasso Sea for the European eel - is currently under discussion based on recent molecular studies and may need to be critically revised (see section “the multiple spawning areas within and outside of the Sargasso Sea). Schmidt also concluded that the American eel spawned in an overlapping area to the west, but he had records of only 22 larvae < 10 mm long (Schmidt 1925). Although there are substantial weaknesses to Schmidt's claim (Boetius and Harding 1985) and despite the limitations of his data, Schmidt's conclusions about eels life history are essentially correct and the Sargasso Sea appears to be the primary spawning area for most North-Atlantic eels (American and European). Johannes Schmidt also stated that the peak of European eel spawning was in April and that the spawning area is centred to the Northeast of the spawning area of the American eel, which has its spawning peak in February (Schmidt 1925). The times and areas of eel spawning have been supported and refined through recent analyses of the available historical data by Boetius and Harding (1985), Kleckner and McCleave (1982, 1985) and McCleave et al. (1987). Ichthyoplankton surveys conducted by a group led by McCleave (USA) and a group led by Tesch (Germany) in the 1980s expanded the number of leptocephali < 10 mm collected at sea (Tesch 1982; Schoth and Tesch 1982; Wippelhauser et al. 1985; Castonguay and McCleave 1987; McCleave and Kleckner 1987; Kleckner and McCleave 1988; Tesch and Wegner 1990). The collection now comprises more than 700 American eel leptocephali and more than 1600 European eel leptocephali <10-mm long (McCleave et al. 1987). All catches of American eel leptocephali < 7 mm total length (188 specimens) were obtained within a broad ellipse extending eastward from the Bahamas to about 58o W longitude. All catches of European eel leptocephali < 7 mm long (226 specimens) were obtained within a narrow overlapping ellipse. The distribution of American

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and European eel larvae < 7.5 mm TL is limited to the north by the boundary between warm saline surface water of the southern Sargasso Sea and a mixed convergence zone of water. Larvae < 7 mm TL are accepted as an indicator of spawning during the preceding three weeks, which is based upon assumed length at hatching and a growth curve developed from artificial maturation experiments in the laboratory (Yamamoto and Yamauchi 1974; Yamauchi et al. 1976). Based on all these observations, we now know that the European eel spawns primarily from March to June within a narrow ellipse whose long axis extends east-west from approximately 48o to 74o W longitude between 23o and 30o N latitude and that the American eel spawns primarily from February to April within a broader oval between approximately 52o and 79o W longitude and 19o and 29o N latitude (McCleave et al. 1987). So spawning of the European and American eel species is partially sympatric in space and time (McCleave et al. 1987). Continental separation of the two species is probably ensured by initial distributional bias from partially allopatric spawning and by different developmental rates (Tesch, 2003). Differences in vertical migration between the leptocephali of the two eel species can partly explain how Anguilla rostrata detrains from the Gulf Stream to invade the North American coast, while Anguilla anguilla presumably stays in the stream on its way to Europe (Castonguay and McCleave 1987). Social interactions and the existence of a species-specific pheromone (McCleave 1987) may help prevent interbreeding. Our observations of spawning behaviour in hormone treated European eels in a 4,000-liter aquarium strengthen the probability that spawning is triggered by pheromones (Section “Spawning behaviour and reproduction”). Based on the distribution of newly hatched leptocephali, it is believed (Kleckner et al. 1983; McCleave and Kleckner 1985; McCleave et al. 1987) that adults of both species spawn in, and to the south of, a persistent, meandering, near-surface frontal zone that stretches east- west across the Sargasso Sea (Voorhis and Bruce 1982). This is the so-called subtropical convergence zone (STCZ), a region where the colder water of the northern Sargasso Sea meets the warmer water of the southern Sargasso. This natural boundary divides the surface waters of the Sargasso Sea into distinct northern and southern water masses (Katz 1969; Voorhis 1969; Kleckner et al. 1983). There are sharp fronts in the STCZ, with shingles of 100-300 km length, separating water masses in the subtropical frontal zone. These fronts act as a boundary for many organisms and some feature of the frontal zone or the southern waters, such as odour or temperature, may serve as signals to migrating eels to cease migrating and spawn (Kleckner et al. 1983;

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McCleave 1987; McCleave et al. 1987). Earlier work of a German group corroborates these results (Schoth and Tesch 1982; Wegner 1982). For Anguilla larvae, leptocephali are much more abundant on the south face of the front that separates the two general water masses in the STCZ (Kleckner and McCleave 1988; Tesch and Wegner 1990). Greater abundances of larvae from other families of shelf eel species (Chlopsidae, Congridae, Moringuidae, Muraenidae and Ophichthidae) and other fish species have been found at or south of fronts in the STCZ of the Sargasso Sea (Miller 1995). It is hypothesised that differences in species composition are caused by a marked decrease of primary production south of the front (Kleckner et al. 1983; Miller 1995). This reduction in primary productivity, combined with the seasonal stability of this layer, may provide a variety of persistent olfactory cues, distinct from those of the northern water mass, providing olfactory signals to eels returning to spawn after many years in freshwater. It is possible that the homing mechanism of adult eels may be based on a similar mechanism to that found in Atlantic salmon, imprinting on odours and tastes of the waters of the southern Sargasso Sea. For sexually immature eels it has been demonstrated that their olfactory senses are highly developed. They are capable of detecting chemical compounds (such as β-phenylethanol) at dilutions as low as 1:2.85 x 1018 (Teichmann 1959). In an experiment, the estuarine migration of anosmic and control silver-phase American eels was examined during spawning migration in fall. Control eels moved more rapidly, using tidal properties to leave the estuary. In contrast anosmic eels took a longer time to leave the estuary and they were unable to use tidal stream transport for movement out of the estuary (Barbin et al. 1998). From these observations it can be concluded that olfaction plays an important role at (initial) migration in adult eels. Another possibility is that a temperature gradient in the surface waters of the frontal zone as high as 2o C per km (Voorhis 1969) could act as a triggering or orientation mechanism. From our swim experiments we obtained data regarding the swim potential of eels (Section “Swimming capacity of silver eels”). Thus we can assume that an eel with a size of 1 m and swimming speed of approximately 1 body-length (BL) per second could experience a temperature difference less than 0.002o C per second. Based on telemetry observations of diurnal migration patterns of migrating silver eels with correspondingly larger temperature fluctuations, it seems unlikely that temperature acts as orientation cue (Tesch 1978, 1989). Recently, we studied the orientation of yellow (non-migratory) female eels in a freshwater pond to the earth’s magnetic field by means of microchips injected into their muscle (van

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Ginneken et al. 2005b). Detectors for microchips were mounted in tubes placed in the pond to determine if eels orientated themselves with respect to earth’s magnetic field. There was a seasonal component in the orientation mechanism, with a significantly lower preference for specific orientation in summer compared to fall. A preference for tubes orientated in a south- southwest direction (the direction of the Sargasso Sea) in fall suggested orientation to the earth’s magnetic field may play a role in migration in eels (van Ginneken et al. 2005b).

3) Leptocephali transport The migration of leptocephali from the area of the Sargasso Sea to the continental shelves and coastal water is very complex and cryptic, foremost because of an incomplete understanding of elements of the physical environment which contribute to variability in ocean transport like recirculation, meandering, eddy formation and tides (McCleave 1993). Secondly, most leptocephali undergo daily and ontogenetic vertical migrations (Schoth and Tesch 1984; Castonguay and McCleave 1987). The latter term indicates that leptocephali undergo changes in vertical distribution with age. Thirdly, we do not know whether the transport of European leptocephali larvae across the Atlantic is based on passive and/or active processes, depending on the larval developmental stage (see further this section). Schmidt (1925) provided little information on vertical distribution of leptocephali of the American and European eel in the Sargasso Sea. He stated only that larvae 7-15 mm long were found between 75 and 300 m deep, whereas 25 mm larvae were found in the water layer between the surface and 50 m. Studies performed more recently, indicated that Anguilla leptocephali < 5 mm long did not exhibit a diel vertical migration, as they were distributed between 50 m and 300 m both by day and night (Castonguay and McCleave 1987). Anguilla of the length range 5-19.9 mm mostly occurred between 100 m and 150 m by day and between 50 m and 100 m by night (Castonguay and McCleave 1987). While Anguilla > 20 mm were found deeper than Anguilla < 20 mm by day, between 125 m and 275 m, and mostly between 30 m and 70 m by night (Castonguay & McCleave 1987). This pattern of migration at shallow warm depths at night and diving to deeper, colder depths during day (probably to avoid high light intensities) has been confirmed in another study west of the European continental shelf (Tesch 1980). In this study the depth preference of leptocephali during daylight was 300-600 m, and at night 35-125 m (Tesch 1980). Based on these diurnal patterns of larvae distribution it can be concluded that larvae < 5 mm have no active transport mechanism while from a size of > 5 mm on active movement may play a role. Also based on morphologically parameters, active swimming of larvae < 5 mm can be excluded, because they are so primitive at hatch

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that an effective swimming mechanism can be excluded (Yamamoto and Yamauchi 1974; Yamauchi et al. 1976, Pederson 2003, Palstra et al. 2005). Therefore, it is assumed that Anguilla larvae < 5mm were probably spawned no more than seven days prior to capture and the depth of catch can be indicative of the spawning depth of the adults. The water of the Sargasso is 5 km deep, but spawning probably takes place in the upper few hundred meters. This is not only based on the depth of catch of < 5 mm larvae, but also on the release of hormone treated European and Japanese adult female eels with telemetry transmitters (see section 7). Although the circulation patterns and oceanic currents are complex and poorly understood, some information is available on the transport of leptocephali larvae out of the Sargasso Sea area with movements toward coastal areas. Discontinuities in the assemblages of Anguilla within and among transects suggest that convergence of surface water toward fronts in the STCZ may concentrate leptocephali close to the fronts and that frontal jets may transport leptocephali eastward (Miller and McCleave 1994). The size distributions of leptocephali suggest that gyres in the south-western Sargasso Sea, an Antilles Current, and the Florida Current north of the Bahamas are routes of exit for anguillid eels. Most leptocephali enter the system north of the Bahamas rather than through the Straits of Florida or island passages (Kleckner and McCleave 1982; McCleave and Kleckner 1985). A previously hypothesized persistent Antilles Current sweeping north-westward along the eastern edge of the Bahamas is no longer believed to exist (Olson et al. 1984). The most important transport mechanism of leptocephali westward toward the northern Bahamas is a gradual advection mechanism. The other transport pathway, which is of minor importance, is southward toward Hispaniola on circulation mechanisms described by Olson et al. (1984). Most of the juvenile eels entering European waters are European eels, but less than 1 per cent are American eel, judged by vertebral counts (Boetius 1980). It is not known how many European eels colonise the American continent. Given the overlap in spawning period and spawning grounds of American and European eels (McCleave et al. 1987; Tesch and Wegner 1990) a substantial fraction of leptocephali of both must be subjected to similar advective processes in the North Atlantic. Therefore, it is unclear what mechanism is the basis for the split between the two species distributing only such a small fraction of leptocephali to habitats outside of their continent of origin. It is possible that there is a clear genetically determined active choice of the water currents used by the larvae (Kleckner and McCleave 1985). Another possibility is a strict, genetically determined period of metamorphosis (Power and McCleave 1983; McCleave 1993; Cheng and Tzeng 1996), which ultimately brings the

240 Annex 1 larvae into contact with the different currents flowing to the American or European continent. Clear differences in metamorphose time and capabilities between the two species have been reported (Kleckner and McCleave 1985, van Utrecht and Holleboom 1985). American eel leptocephali may become developmentally capable of undergoing metamorphosis after 6-8 months and remain viable for 4-6 months (Kleckner and McCleave 1985). In contrast, European leptocephali become capable of metamorphosis only after about 18 months, but remain viable for several years (van Utrecht and Holleboom 1985). New knowledge about timing of metamorphosis is available in Lecomte-Finiger, 1994 and Arai et al, 2000. According to Lecomte-Finiger (1994) the mean age of glass eel ranged from 190-280 days. The calculated growth rate was 0.26-0.30 mm per day. Thus, European eel larvae spend less than one year in transatlantic migration (Lecomte-Finiger 1994) in contrast to the earlier estimated period of 2-3 years (Schmidt 1922). Arai et al. (2000) gave more detailed information based on Otholith microstructure and microchemistry. Otholith increment width markedly increased from age 132 to 191 d (156 ± 18.9 d; mean ± SD) in A. rostrata and 163 to 235 d (198 ± 27.4 d; mean ± SD) in A. anguilla. The duration of metamorphosis was estimated to be 18 to 52 d from otholith microstructure, for both species studied. Age at recruitment were 171 to 252 d (206 ± 22.3 d; mean ± SD) in A.rostrata and 220 to 281 d (249 ± 22.6 d; mean ± SD) in A. anguilla. (Arai et al. 2000). Currently there are two theories about larval transport from the spawning area to the coastal habitats of different continents. One theory suggests a passive multi-year and variable oceanic transport (van Utrecht and Holleboom 1985; Guérault et al. 1992). The other theory states that larvae transport is an active process of short duration, including the time of metamorphosis of European eels of only 7-9 months (Lecomte-Finiger 1994, Arai et al. 2000 see also section “The location of the spawning areas”). It is difficult to choose between the multi-year passive and active larvae transport theories due to problems that arise from the interpretation of glass eel otholiths. There are conflicts about the accuracy of ageing glass eels using SEM (Scanning Electron Microscope) otholithometry. In general it is suggested that there is a relationship between otolith increment deposition and somatic growth. This method was used by Lecomte-Finiger (1994) to state that migration of glass eels from the Sargasso Sea was an active and not a passive process. However, in practice the matter is more complicated. A first methodological problem is that light microscopy can not resolve objects separated by less than 0.2 μm (Campana and Neilson 1985), so they cannot be used to count zones in the so called “B-type” otholiths. B-type otholiths are probably from slow growing

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animals without clear regular incremental separations. Increments of around 1.9 μm are found in normal growing animals with so-called “A-type” otholiths (Umezawa and Tsukamoto 1991). A second problem is that despite the close relationship between increment counts and body growth, other factors also may affect the size and deposition of otholith increments, such as water temperature, feeding ration, feeding frequency, starvation and photoperiod (for references see Umezawa and Tsukamoto 1991). Catadromous fish species such as eels and their larvae may experience enormous differences in food supply, temperature, salinity etc. during their seaward migration. Therefore information about growth rates for leptocephali of both American and European eel has to come from growth studies under optimal standardised conditions. Luckily, Pedersen (2003) and the Leiden research group (Palstra et al. 2005) have succeeded in the production of leptocephali of the European eel allowing the development of clinical/assessment of growth rates under experimental conditions.

4) The possibility of multiple spawning areas within and outside of the Sargasso Sea. The hypothesis that all European eels migrate to the Sargasso Sea for reproduction and constitute a single randomly mating population, the so-called panmixia theory, is generally accepted. However, based on field observations (Grassi 1896; Bast and Klinkhardt 1988; Lintas et al. 1998), morphological parameters, such as the total number of vertebrae (Boetius 1980; Harding 1985), and recent molecular work (Lintas et al. 1998; Bastrop et al. 2000; Daemen et al. 2001; Wirth and Bernatchez 2001; Maes and Volckaert 2002) there are some indications that the European eel population is genetically diverse, pointing to discrete spawning populations. Nevertheless, the latest extensive genetic survey indicated that the geographical component of genetic structure lacked temporal stability, emphasising the need for temporal replication in the study of highly vagile marine species (Dannewitz et al, 2005). Hence, indications for as well one single as several discrete spawning sites have been provided in the last century, which will be discussed in this section.

a) Classical arguments In the 1960’s, Tucker (1959) and D'Ancona (1960) hypothesized that eel spawning areas could be located in the Mediterranean close to the Strait of Messina (a 2000 meters deep- water body in the south of Italy). This assumption was based on the lack of any catch of a migrating maturing eel in the narrow Strait of Gibraltar despite considerable research efforts

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(Ekman 1932). In contrast, migrating silver eels have been caught in the Sont (the narrow Sea Strait of 4,5 km width in Denmark connecting the North Sea and the Baltic Sea) and the Strait of Dover (Tucker 1959). Additionally, only one maturing eel with a Gonado-somatic Index (GSI) of 10 has been caught west of Morocco, close to the Azores (Bast and Klinkhardt 1988), which may point to the existence of another spawning area located west of Morocco. However, conclusions based on sporadic catch data remain highly speculative and to date no serious attempts have been made to catch eels in the open Atlantic (see section “Tracking silver eel migration”).

There are several further ‘traditional’ arguments against the single spawning site theory: a) Grassi and Calandruccio discovered in 1896 in the Strait of Messina leptocephali larvae of 50 mm, which they ascribed to the larval stage of the European eel (Grassi 1896). b) Some authors reported the presence of adults with enlarged eyes (an indication for advanced sexual maturity) in the Strait of Messina (Lintas et al. 1998). c) A re-evaluation of the total number of vertebrae (TNV) in European eel samples collected by Johannes Schmidt demonstrated that Schmidt's claim of homogeneity of the eel population and a unique spawning location was an overstatement (Harding 1985). The number of vertebrae increased on a North-South latitudinal gradient along the Atlantic coast. In the Mediterranean, a significantly heterogeneous distribution in TNV was observed, without any apparent geographical cline. Harding (1985) suggested at least two, possibly three, distinct groups, each with their own distribution of length and total numbers of vertebrae. Environmental influences in the early life phase of larvae, including their origin in separate parts of the spawning area and different migration routes to the European coasts could, however, result in similar trends (Harding 1985). d) Very young glass eel have been observed along the Atlantic coast, from Morocco to the Netherlands and in the Western Mediterranean (Lecomte-Finiger 1994). This may be indicative of spawning areas west of Morocco, closer to the European continent than the Sargasso Sea.

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On the other hand, “traditional’ arguments in favour of the single spawning site theory include:

a) No spawning adults have ever been observed in the Mediterranean Sea (note: this is also the case in the Sargasso Sea). b) Eels are rarely observed in the Black Sea, which is not expected if separate eel populations would spawn in the Mediterranean Sea. c) The number of vertebrae of eels from the Atlantic corresponds to that of eels from the Mediterranean (Tesch 2003). d) The Mediterranean contains only leptocephali larvae > 60 mm long. e) These larvae become larger from the west of the Mediterranean to the east. f) Coherence in recruitment patterns gave no evidence for any subdivision of the European eel stock (Dekker 2000).

b) Molecular arguments Molecular data have also provided both evidence supporting and rejecting the Panmixia hypothesis using various genetic markers. They will be reviewed chronologically to provide an overview of the shifts in ideas, along with the continuous development of new molecular markers.

Early population genetic studies, based on observed differences in transferrines and liver esterases, claimed that European eel populations differed between several continental European locations (Drilhon et al., 1966, 1967; Drilhon and Fine 1968; Pantelouris et al., 1970), suggesting that eels in the south-eastern part of the Mediterranean formed a separate group and reproduce in this area. This supported the theory of discrete populations, although differential selection was also proposed as a possible explanation (Pantelouris et al. 1970, 1971). However, the conclusions of most allozyme-based studies from the 1960s have been re-evaluated and rejected on methodological grounds (Koehn, 1972). Later allozymatic studies failed to detect obvious spatial genetic differentiation (de Ligny & Pantelouris, 1973; Comparini et al., 1977; Comparini & Rodinò, 1980; Yahyaoui et al., 1983).

Studies based on mitochondrial DNA initially provided only limited insights into the geographical partitioning of genetic variability in European eel, mainly because of the very high number of haplotypes in the D-loop region and the expected recent timescale of

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intraspecific differentiation (Lintas et al., 1998). The study of Lintas et al. (1998) supports the genetic homogeneity of the European eel population. They sequenced the 5’ end the mitochondrial D-loop of 55 eels caught at different European locations, known to show high levels of nucleotide substitutions among teleosts (Lee et al. 1995). Nevertheless, Lintas et al. (1998) found so little DNA differentiation among European eel individuals from distant geographical locations, that they suggested all European eels being derived from a common genetic pool. A recent study by Bastrop et al. (2000) confirmed this result based on 16sRNA sequences. Although the European eel population is genetically more diverse than the American eel population (Avise et al, 1986, Bastrop et al, 2000) and the genetic homogeneity of the European eel seemed beyond dispute according to these recent molecular DNA studies (Lintas et al. 1998; Bastrop et al. 2000), the possibility remained of multiple spawning areas. Lintas et al. (1998) hypothesised two situations in which the European eel would remain genetically homogeneous with the existence of several discrete spawning areas:

1) A partial reproductive isolation with some gene flow between eels from the Mediterranean and the Sargasso Sea. 2) Other spawning sites than the Sargasso Sea with mixing of larvae originating from different breeding areas.

Panmixia in the European eel became thus widely accepted until three independent recent genetic studies reported evidence for a weak but significant population structure (Daemen et al., 2001; Wirth & Bernatchez, 2001, Maes and Volckaert, 2002). New indications of the non-random distribution of haplotypes were reported using the less variable cytochrome b mtDNA marker (Daemen et al., 2001). European eel populations exhibited much lower haplotype diversity at the cytochrome b locus compared to the 5’ end of the D-loop (Lintas et al., 1998). The genetic variation observed at the cytochrome b locus was nevertheless high (17 haplotypes in 107 eels), with two central haplotypes in the haplotype network and a significant latitudinal clinal pattern of cytochrome b haplotypes fitting an isolation-by- distance model. Further, Daemen et al. (2001) detected a weak but significant genetic differentiation among the British/Irish, Atlantic, Moroccan, Italian and Swedish Baltic populations, respectively, using five nuclear microsatellite loci. In a later study, Wirth and Bernatchez (2001) also identified weak but highly significant genetic structure in the European eel population among 13 samples, based on seven microsatellite loci, reporting evidence for isolation-by-distance (IBD) (Figure 4b). Finally, Maes and Volckaert (2002)

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reported clinal genetic structure and IBD in the European eel population using 15 allozyme loci and identified three distinct groups: Northern Europe, Western Europe and the Mediterranean Sea.

Figure 4: Genetic evidence based on microsatellites in favour of and against the Panmixia hypothesis using a) combined geographical and temporal (Dannewitz et al, 2005) or b) exclusively geographical (Wirth & Bernatchez, 2001) samples across Europe.

Results from the former genetic studies pointed to the existence of a genetic mosaic in the European eel, consisting of several isolated spawning groups. According to Wirth and Bernatchez (2001), and Maes and Volckaert (2002), in theory three models can explain the rejection of the panmixia hypothesis:

a) There is one common spawning area, but there is a temporal delay between the arrival of adult eels originating from different latitudes. b) There is one reproductive area used by different populations where different sea currents carry the leptocephali back to their parent’s original freshwater habitat. c) There is only one shared spawning area where assortative mating occurs and larval homing to parents’ habitat takes place using an unknown mechanism.

Finally, the most recent and extensive genetic study on European eel increased significantly the geographical sampling (42 sites) and included crucial temporal replicates (at 12 sites) into their analyses to check for consistency in the observed spatial pattern (Dannewitz et al.

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2005). Surprisingly, no stable spatial genetic structuring was detected anymore, while temporal variance in allele frequency exceeded well the geographical component (Figure 4a). Possible sampling bias due to life stage mixing and a lower effective population size than expected could explain these conflicting results (Dannewitz et al. 2005).

In summary, nuclear and mitochondrial DNA data provided evidence for a subtle heterogeneous European eel population, with a minimal geographical component across Europe, but with most genetic variation being present between temporally separated populations. Such results reflect the high variance in reproductive success in marine species in general, inducing small and large-scale temporal changes in genetic composition between cohorts (Dannewitz et al, 2005; Maes, 2005; Pujolar et al., 2005b).

c) Evidence of a single or multiple spawning sites in other Anguilla spp. Similar results of lack of differentiation were observed in several other eel species. The American eel (A. rostrata) showed no evidence for a geographical subdivision, with the exception of clinal allozyme variation putatively imposed by selection (Williams et al. 1973; Koehn and Williams 1978; Williams and Koehn 1984; Avise et al. 1986, Wirth & Bernatchez, 2003). These data suggested that Anguilla rostrata is genetically homogenous, forming a single randomly mating population. In the Japanese eel (Anguilla japonica), no evidence was found of genetic structure over large geographic areas in studies based on mitochondrial DNA (Sang et al., 1994; Ishikawa et al., 2001), but clinal variation was observed at allozymes (Chan et al., 1997). In A. australis and A. dieffenbachii, an allozyme based study showed a signal of differentiation between recruiting and resident populations (Smith et al., 2001). In the giant mottled eel (A. marmorata), even several genetically isolated populations could be detected using mtDNA (Ishikawa et al., 2004). Intra-specific divergence was of the same level as the lowest inter-specific divergence in the genus Anguilla between the North-Atlantic eels or between the sub-species of A. bicolor. The distribution pattern of five populations was closely associated with the water-mass structure of oceans and major current systems. This observation suggests that present population differentiation in A. marmorata might have resulted from the establishment of new population specific spawning sites in different oceanic current systems as the species colonised new areas (Tsukamoto et al., 2002; Ishikawa et al., 2004).

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d) Evolutionary consequences of the European eel’s life-history traits After consideration of all arguments from the traditional and molecular studies, we are able to summarize and extend some conclusions in favour or against the panmixia hypothesis. Several life cycle characteristics in the European eel may or may not contribute to genetic structuring: a) Age at maturity is highly variable, ranging from 6 to 50 years in females (Poole and Reynolds, 1996) over a latitudinal gradient. In Northern Europe the mean age at maturation of females can range from 12-20 years (or older), while in Southern Europe it is 6-8 years (Tesch 1977). If there is a temporal segregation of populations in Europe by age (latitudinal gradient), adults from various continental locations may mate assortatively in the Sargasso Sea and may be able to maintain their integrity throughout the arrival waves (Maes and Volckaert 2002). Hence, the population in Europe may consist of an admixture of subpopulations. The development and maintenance of such a structure nevertheless requires temporal and/or spatial separation in the Sargasso Sea of spawning adult eels originating from different locations in Europe. This has to be followed by a non-random return of larvae to their parents’ freshwater habitat through active swimming, seasonal changes in hydrodynamics or different pathways of the Gulf Stream (Wirth & Bernatchez 2001; Maes & Volckaert 2002). Dannewitz et al (2005), however, provided evidence in favour of panmixia (no stable isolation-by-distance (IBD)), indicating that any geographical component visible in a specific year would be inevitably lost due to the environmental dependency of age at maturity and the subsequent extensive mixing of formerly distinct spawning cohorts. b) The different life history of males and females also leads to different maturation patterns and timing. Males tend to mature at a size of around 40 cm and at an age of 3-4 years, while females mature at a size of > 60 cm and at an age of 6-8 years (or older). Such maturation pattern complicates the potential to build up and maintain a stable genetic structure, because of the latitudinal bias in sex ratio (Tesch, 2003). Although different ages at maturity between sexes do not constitute a restriction to develop and maintain population structure, a lack of geographical differentiation in favour of temporal differences may break up any temporal differentiation between cohorts distributed “randomly” over the European continent. Studies using mitochondrial DNA (mtDNA), which is inherited only maternally, did not show any geographical clustering (Avise et al. 1986; Sang et al., 1994; Lintas et al, 1998), pointing to the lack of power of this marker at the temporal scale studied or an unusual pattern of female mediated gene flow. The first hypothesis seems most plausible and could be indicative for a

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recent post-Pleistocene divergence pattern. A more thorough analysis of mtDNA markers on many individuals would probably be needed to fully assess the potential of this marker, as subtle differences in marine species are more expected to occur at the haplotype frequency (quantitative) level than the haplotype distribution (qualitative) level. c) Adult eels exhibit differential migration departure times during spawning season, not only between populations in a North-South gradient but also between the sexes. For the smaller males it takes a longer time period to cover the distance of 6,000-km to the Sargasso Sea. Assuming a swimming speed of 0.5 Body-Lengths per second, a 80 cm female would reach the Sargasso Sea in 174 days, while this would take for a 50 cm male 278 days. Males usually depart 1-2 months earlier than females (Usui 1991, Tesch, 2003). In the Netherlands, the seaward migration of silver males starts in August while the first females start migrating in September or October (Usui, 1991). This protracted spawning period will increase the chance for overlap between possibly differentiated populations, although if spawning migration departure is genetically determined, cohort differentiation may be maintained throughout the spawning season. Nevertheless, the differential departure time over a latitudinal gradient and between sexes likely evolved to maximise the chance of group spawning in the Sargasso Sea at the most favourable period (coinciding with the larval bloom). d) The European eel exhibits the largest “migration” loop of all Anguillids (Tsukamoto et al. 2002). The potential breeding area is 5.2 x 106 km2, so there can be a great deal of separation in space and time among spawning stocks. As long as the question has not been answered why the Sargasso Sea is so unique for eels reproduction, and as long as the exact location has not been confirmed, the total area can be seen as potential breeding grounds. From behavioural observations of spawning eels in aquaria (see section “Spawning behaviour and reproduction”), indication of collective and simultaneous spawning have been found; pheromones may play an important role in finding partners (McCleave 1987) (see section “Spawning behaviour and reproduction”). Hypothetically, adults from various continental locations could mate assortatively in sub-areas of the overall breeding grounds attracted to each other by specific odour. This separation mechanism may lead to a genetic mosaic consisting of isolated populations, although the temporal persistence of this mechanism remains questionable (Dannewitz et al., 2005; Maes, 2005). e) The possibility to detect separate discrete spawning adults in the Sargasso Sea can be blurred due to the subsequent mixing of offspring during their journey to Europe. Random larval dispersal to the continent may mask active mechanisms of genetic structuring. In eels, however, active migration has been shown to distribute larvae along a latitudinal gradient

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following age/length (Lecomte-Finiger, 1994; Arai et al. 2000). Additionally, both North- Atlantic eel species show a strong directional migration to each continent, supporting the potential for active orientation of leptocephali larvae. Further indications for non-random larval dispersal are the observation of hybrids between American and European eels in Icelandic eel populations. Hybrids between both species, which are found almost exclusively in Iceland, may exhibit a genetically defined intermediate migrational behaviour (Avise et al. 1990; Maes, 2005), with an intermediate developmental time. If randomly distributed across Europe, hybrids would have to be found in the Western British Isles, first passed by North- Atlantic currents. f) Finally, due to the unpredictability of the oceanic environment, marine species often show a very high variance in reproductive success and will evolve a strategy to maximize their offspring’s survival (Hedgecock, 1994). In eels, considering their extremely long trans- oceanic migration as adult and larvae, a protracted spawning period and random mating may be the best strategy to maximise the chance of reproducing in favourable conditions. Although seasonal reproduction of subpopulations could occur, the chance of complete reproductive failure of certain groups is real (mismatch with algae bloom), endangering the survival of the species in the long term (Hedgecock, 1994; Maes, 2005; Pujolar et al., 2005b).

e) Future genetic research perspectives in the European eel Conclusions drawn from molecular studies are a crucial tool to infer the panmictic status in the European eel. Considering the contrasting outcomes from recent molecular studies (Wirth & Bernatchez, 2001 vs Dannewitz et al., 2005; Figure 4), future research could focus on several of the following directions, to help clarify European eels evolution:

• The standardized small-scale analysis of recruiting juveniles may provide additional answers about the spatio-temporal partitioning of genetic variation and the presence/absence of a genetically determined spawning time (Pujolar et al., 2005b). • The analysis of long –term time series of historical material may increase the confidence of genetic estimation of genetic population sizes. A first step would be the use of aged adults, so that back calculations till 30-40 years ago can be performed. More importantly, to assess the influence of heavy fisheries and yearly/decadal fluctuating oceanic conditions, the analysis of historical material covering the last century is urgently needed. This is now possible due to newly developed genetic techniques for ancient DNA and will enable the

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reliable calculation of a pre- and post industrial fishery genetic population size. This knowledge is of crucial importance to preserve genetic variation, known to correlate with fitness components in eel (Maes et al, 2005; Pujolar et al., 2005a) and to define sound management issues. • Although intraspecific genetic structure is very subtle in many eel species, neutral genetic variation might well underestimate adaptive variation over a broad environmental range. The development and study of novel markers under selection (such as Expressed Sequence Tags (ESTs) and Single Nucleotide Polymorphisms (SNPs) in candidate genes) would enable the detection of genetic variation underlying environmentally dependent fitness traits. SNPs are considered the markers of the future, due to their unambiguous scoring (compared to microsatellites), short fragment size (suitable for ancient DNA), neutral/adaptive characteristics and uniform polymorphism across the genome (Syvänen, 2001). • The current fishery pressure on the European eel stock is mostly due to the lack of artificial reproduction (but see Palstra et al., 2005 and references therein). Since 30 years, researchers have been unable to produce economically profitable quantity of eels in aquaculture. Integrating additional oceanic knowledge into management strategies, together with the reduction of fisheries, might help define sustainable management issues, until artificial reproduction is successful.

The European eel has been studied for over hundred years and hypotheses concerning its population structure were tested using newly developed techniques every time they appeared. Nevertheless, the black box remains tightly closed for researchers. Many factors of its catadromous life-strategy increase the chance of panmixia, such as the variable age at maturity, the highly mixed spawning cohorts, the protracted spawning migration, the sex biased latitudinal distribution and the unpredictability of oceanic conditions. Nevertheless, several active components induce the chance for population divergence, such as assortative mating behaviour, the segregation of both North-Atlantic species in the Gulf Stream, active trans-oceanic larval migration, the presence of hybrids mainly in Iceland and the extremely large migration loop of the European eel compared to other species. In this review of traditional and genetic knowledge, it became clear that a geographical component, if existing, is almost invisible. On the other hand, genetic data supports strong temporal variation between and within years/cohorts possibly as a consequence of large variance in adult contribution and reproductive success (Dannewitz et al, 2005; Maes, 2005; Pujolar et al.,

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2005b). Oceanic forces are likely to represent one of the main actors in the observed temporal variation. The present climatic oscillations combined with the significance of oceanic forces in marine species prompts to the urgent assessment of temporal stability of the European eel stock, combining genetic, population dynamics and oceanic data. Only by tracking migrating adults and genetic monitoring their offspring through time, a reliable assessment of the factors influencing the population structure of the European eel will be possible.

5) Are European and American eels sharing the same spawning grounds?

There are only two species in the North-Atlantic Ocean, the European (A. anguilla) and the American eel (A. rostrata). Based on the number of vertebrae, the American eel (vertebrae ranging from 103-110, mean 107.1) can be distinguished from the European eel (vertebrae ranging from 110-119, mean 114.7) (Boetius 1980). It is assumed that the spawning area of both eel species is located in the Sargasso Sea (Schmidt 1935; Ohno et al. 1973; Comparini and Rodino 1980; McCleave et al. 1987; Tesch and Wegner 1990). Several scenarios have been proposed for their origin, based on fossil records, plate tectonics, paleo-currents and a standard fish molecular clock. A first scenario is the dispersal of ancestral organisms through the Tethys Sea that separated 70 million years ago Laurasia (North-America and Eurasia) from Gondwana (South America, Australia, Africa and India). Along this sea, dispersal was possible through westerly paleocircumglobal equatorial currents (Aoyama & Tsukamoto, 1997; Aoyama et al., 2001). Aoyama et al. (2001) suggest that Anguilla speciation started 43.5 Mya and that the North-Atlantic eels speciated some 10 Mya. Although such results were partially confirmed by another study (Bastrop et al., 2000), Lin et al. (2001), using a much larger fragment of the mitochondrial genome (cytochrome b and 12sRNA), proposed that the genus Anguilla speciated much more recently, some 20 Mya. This study hypothesised that the Atlantic eels colonised the North Atlantic through the Central American Isthmus (Panama) and speciated only some 3 Mya. Although these authors used a longer fragment and their speciation estimates are much more congruent with the accepted molecular clock, some incongruence remained. The absence of any eel species on the West coast of North-America or South America and the large phylogenetic distance with A. japonica, who should under this scenario be the ancestor of the North-Atlantic eels, suggest that the radiation events are much more complicated than expected using present day current and tectonic knowledge. A recent study analysing the complete mitochondrial genome gave additional support for the first hypothesis’ dispersal route, but for the second

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hypothesis’ speciation time (Minegishi et al., 2005). Speciation started 20 MyA and formed two main clades, the Atlantic-Oceanian group and the Indo-Pacific group. The present day geographical distribution does not seem to follow phylogenetic relationships anymore in the former, but does so in the latter group (Minegishi et al., 2005). Nuclear data might be the next step to clarify these ambiguities. These results also confirm the instability of morphological characters to discriminate the evolutionary relationships between Anguilla species, even after a thorough revision (Ege, 1939; Watanabe et al., 2004). The divergence between both North-Atlantic species has been under discussion for decades. Tucker (1959) claimed that differentiating meristic characters (number of vertebrae) were under ecophenotypic selection during the transoceanic migration. The European eel would be the offspring of the American eel. Tucker (1959) suggested that the European eels do not participate in reproduction, because the distance to the Sargasso Sea was considered too far. Later work, based on variation at hemoglobin, transferrins and allozymes however, confirmed the two species status (Fine et al., 1967; Drilhon et al., 1966, 1967; Drilhon and Fine 1968, de Ligny & Pantelouris, 1973; Comparini & Rodino, 1980; Comparini & Scoth, 1982). Also two studies using specific proteins from respectively muscle and eye lens tissue indicated that the two Atlantic eel species have diverged far enough to have accumulated distinctive genes. One study was based on electrofocusing methods using polyacrylamide gels for muscle protein differences (Jamieson & Turner 1980). Another study used eye lens proteins as genetic markers using patterns of isoelectric point variation (Jamieson & Teixeira 1991). The allozyme locus MDH-2* exhibits a nearly fixed difference between both species, although Williams & Koehn (1984) questioned the taxonomic reliability based on only one enzymatic locus. A mitochondrial DNA RFLP study showed conclusive results, separating both species with high confidence at 11 out of 14 restriction endonucleases, although the two North-Atlantic species exhibited the lowest genetic distance reported between Anguilla species (Avise et al., 1986; Tagliavini et al., 1995; Aoyama & Tsukamoto, 1997; Ishikawa et al., 2004). The two Atlantic eel species cannot unambiguously be discriminated based on cytogenetic criteria like CMA3 staining, and FISH (fluroresence in situ hybridization (Salvadori et al. 1995)), or C-and G-banding (Salvadori et al. 1996). Another study assessed the North-Atlantic eel speciation process using jointly distributed parasites (Marcogliese & Cone, 1993). They reviewed the “oceanic” and the “vicariance” hypothesis of Avise et al. (1990), suggesting that the two species diverged either in sympatry through differential currents or through the influence of the ice sheets during the Pleistocene, respectively. In the first hypothesis, eels were supposed to live along a single coast (American or European) and

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disperse through changing currents to the opposite side of the Atlantic, with subsequent assortative mating. The second hypothesis states that the ancestor species had a broad continuous distribution, but split into two groups distributed at each side of the Atlantic under the influence of southward Pleistocene glaciations. The vicariance hypothesis seems to be the most likely to explain the present disjunct transcontinental distribution of the parasites in the study, which can only be transmitted horizontally by continental resident individuals living in freshwater (Marcogliese & Cone, 1993). Probably, distinct dispersal patterns during spawning and/or unique spawning grounds provide the basis for the current split between the two species. It is also possible that there is a clear, genetically determined active choice of water currents by the larvae that ultimately brings them to their appropriate continent at different sides of the Atlantic (Kleckner and McCleave 1985). Another possibility is a strict genetically-determined period of metamorphosis (Power and McCleave 1983; McCleave 1993; Cheng and Tzeng 1996), which ultimately brings the larvae into currents directing them to the American or European continent. The North-Atlantic eels have been found to be almost completely reproductively isolated, with a small fraction of genetic exchange. Iceland is mainly colonised by European eels, although a small proportion of eels exhibit a vertebrae number smaller than 110 (Avise et al., 1990).

Even though reproductive isolation is strong, indications for hybrids between European and American eel were detected in two studies. Williams & Koehn (1984) compared the MDH-2* genotypes with the number of vertebrae and concluded that there must be a significant amount of gene flow between both species. Avise et al. (1990) evaluated mitochondrial DNA in addition to nuclear and meristic markers in Icelandic individuals. The data reflected cytonuclear disequilibria, most likely due to ongoing gene flow between both species. The study allowed the detection of pure individuals of both species besides hybrids and a quantification of the American eel material in Iceland (2-4%). Recently, Mank & Avise (2003) reassessed these conclusions with highly polymorphic microsatellites markers. Despite the high resolution and power expected from microsatellite markers (Manel et al., 2002; Anderson & Thompson, 2002), surprisingly no indications for hybridisation were detected (Mank & Avise, 2003). Most likely homoplasy was the main reason for the lack of discriminative power between both eel species. This result prompts for further investigations on the paradigm of complete isolation of European and American eels and reopens the debate of the existence and maintenance of a hybrid zone at more than 6,000 km from the spawning site.

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6) Swimming capacity of silver eels It has long been questioned whether fasting eels have sufficient energy reserves to cover the distance of 5,500 km travelling from the European coasts to the Sargasso Sea. Tucker (1959) had severe doubts whether the European eel would be able to swim across the ocean and suggested that all European eels are the offspring of American eels. Tucker's 'new solution to the Atlantic eel problem' provoked a long debate (D’Ancona and Tucker 1959; Deelder and Tucker 1960), but was finally rejected because a distinction could be made between the two Atlantic eel species based on genetic data (see section “Are European and American eels sharing the same spawning grounds?”). The theory of Tucker (1959), that the European eel is energetically unable to swim 6000 km and would die in the continental waters, can also be rejected by the recent results of energy-balance studies performed in swim-tunnels. Those tunnels were specially developed for long distance migration studies with silver eels at our laboratories. The flow pattern of the tunnels has been evaluated using the Laser-Doppler method (van den Thillart et al. 2004). The oxygen consumption rate was calculated from the oxygen decline after closing the water-inlet with a magnetic valve. This was done daily during a swim period of several months at a fixed time (14.00-17.00 h PM), and oxygen level was recorded minutely on a data-acquisition system. We calculated oxygen consumption from the decline of the oxygen tension (van den Thillart et al. 2004, van Ginneken et al. 2005c ). Results from this study were unexpected. Eels are extremely efficient swimmers due to their elongated flexible body, which is the basis for the characteristic eel-like (anguilliform) mode of locomotion. In one study, nine yellow eels were used with a body weight of 915 g ± 58.4 gram and a length of 74.7 ± 3.4 swimming 0.5 body-length per second at 19 o C. The animals swam 117 days without feeding or resting, day and night. During this period the eels succee- ded in covering a distance of 5533 ± 354 km (figure 5). The loss of weight for the swimming animals over the period of 117 days was approximately 180.3 ± 38.2 g., which corresponds to 19.7 % of the initial total body weight. By two independent methods, oxygen consumption, and carcass composition, we calculated the energy consumed over a six-month swimming period, which we expressed in the COT (gross energy costs of transportation) value. This is the total amount of energy (kJ) it takes to transport one-kilogram body weight over 1-kilometer at a given speed (Schmidt-Nielsen 1972).

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Figure 5: Oxygen consumption of fasting yellow eels from a hatchery (860 ± 81.9 g, 73.1 ± 3.8 cm) during a 6 months period of rest or 6 months of continuously swimming at 0.5 BL/s at 19 ° C. Regression lines: Rest-group: Y=0.0326 X + 25.294; Swim-group: Y=0.0394 X + 54.86. Diamonds: (swimming), circles (resting). (van Ginneken et al. 2005c).

Data from the literature for several sub-carangiform adult fish species, such as salmon, gave COT values in the range of 2.52-2.58 kJ/kg/km (Brett 1973). The oxygen consumption data and carcass analyses gave both COT values of 0.42 and 0.62, respectively. This means that eel swim four to six times more efficiently than non-anguilliform fish such as trout and salmon (van Ginneken et al. 2005c). Analysis of body constituents of the eels at the start and at the end of the experiment revealed that the ratio of all three substrates (lipid, carbohydrate, and protein) remained constant despite significant weight losses. This means that body composition did not change during the six months and that fat, protein, and carbohydrate were used in the same proportion (van Ginneken et al. 2005c). To confirm this difference in swimming efficiency, we allowed eels and trout of the same body weight to swim in our swim tunnels at 18 ° ± 0.3 ° C at comparable body speed in our experimental set up for one week. European eels (n = 5, 155.0 ± 18.3 g, 43.2 ± 3.2 cm) and rainbow trout (Oncorhynchus mykiss, n = 5, 161.5 ± 21.5 g, 24.6 ± 1.0 cm were selected to swim in separate swim tunnels continuously at respectively 0.5 BL/sec (21.5 ± 1.6 cm/sec) and 0.7 BL/sec (17.2 ± 0.7 cm/sec). The eels and trout covered a mean distance of 132.5 ± 12.1 km and 102.8 ± 2.3 km respectively during seven days of continuous swimming.

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Oxygen consumption rates allows us to calculate COT values of 0.68 (eel) and 2.73 (trout) kJ/kg/km. Video films of swimming animals ensured us that the fish were swimming freely and did not benefit from wall effects. This experiment provided two important results: first, the COT value of the small eels is close to that of the larger eels used in the 5,500-km experiment. Second, the observed COT value of the trout in this study is close to previously published values for salmonids (Brett 1973). Hence, we concluded that eels swim around four times more efficiently than salmonids (van Ginneken et al. 2005c). An explanation for this phenomenon may lie in the swimming behaviour and muscle activity patterns of eels, as described by Gillis (1998). At low swimming speed eels do not use anterior muscle, only those located more posteriorly. Thus eels need to recruit only a small percentage of the swimming musculature to swim speeds of 0.5 BL/sec. That eels swim at relatively low swimming speed comes from several animal tracking studies under natural conditions. High speed is not characteristic of the pure anguilliform mode, most reports mention speeds around 0.5-1 BL/sec. For example American eels equipped with pressure sensing ultrasonic transmitters made frequent dives from the surface to the bottom during hours of daylight and darkness at speeds of 0.8-1.1 BL/sec. The maximum rate of ascent was 0.6-0.8 BL.sec (Stasko & Rommel 1974). Migrating Japanese silver eels (Anguilla japonica) have been tracked in the open ocean at a mean speed of 0.48 BL/sec (Aoyama et al. 1999). In a study with yellow- and silver-phase European eels fitted with 300 kHz transponding acoustic tags and tracked by sector-scanning sonar in the western North Sea for 58 h their modest mean swimming speed in midwater was 0.45-0.75 BL/sec (McCleave & Arnold 1999). So all these studies indicate that the swimming speed of migrating yellow and silver eels is between 0.5-1 BL/sec. Some eels used selective tidal stream transport to move northward. (McCleave & Arnold 1999). At this moment we can speculate about the migration of silver eels. Probably they use selective tidal stream transport to cross the continental shelf wherever there are fast and directional tidal streams. Tides also exist in the ocean, so there is a possibility that they also get an assisted passage across the Atlantic if they travel close to the seabed. Otherwise, of course, they may follow prevailing surface currents to get back to the Sargasso. (Personal communication Dr.Geoff Arnold). It would be a challenge to get this information in future studies using archival tags (see Section “Tracking silver eel migrations”).

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An additional advantage for migrating eels under natural conditions, sometimes at depths of 2,000 m (Robins et al. 1979), is the improved efficiency of their oxidative phosphorylation at high pressure (Theron et al. 2000). Although we can speculate about the mechanism which explains the efficiency of anguilliform movement, in future studies, hydrodynamics has to explain how does undulatory swimming work. Therefore two main questions have to be addressed: a) the topic of the muscle design: which muscle arrangement best suits the task of bending the body, b) how does the fish convert muscle power into swimming power (pers.communication: Dr.Ulrike Muller, Wageningen University, The Netherlands). COT values from our study (van Ginneken et al. 2005c) confirm our earlier observation about the swimming capacity of eels suggesting that starving eels are, due to the low energy costs of transport, able to cover long distances (van Ginneken and van den Thillart 2000). In this recent study, we demonstrated that silver eels could swim at very low energy consumption levels, which enables them to use only 40 % of their fat stores for crossing the Atlantic. The remaining 60% of the fat stores are sufficient for gonad development, in theory reaching a GSI of 22 (van Ginneken and van den Thillart 2000). This low energy cost for migration of eels is probably the basis for its uncommon catadromic life cycle with exceptional migratory patterns to their spawning grounds several thousand kilometres away: the European eel travels over 5,500 km to the Sargasso Sea (Schmidt 1923; McCleave and Kleckner 1987; Tesch 1982; Tesch and Wegner 1990 ); the American eel migrates over 4,000 km also to the Sargasso Sea (Castonguay and McCleave 1987; McCleave and Kleckner 1987; Tesch and Wegner 1990); the Australian eel (A. australis) travels over 5,000 km into the Pacific Ocean to spawn (Jellyman 1987); and the Japanese eel (A. japonica) travels over 4,000 km to the Marianna Islands in the Philippines to spawn (Tsukamoto 1992). It can be opposed to the 5,500 km swim study (van Ginneken et al. 2005c) that hatchery eels have an extremely good nutritional condition and the same may not be true for the wild population (Svedäng and Wickström 1997). Therefore, the swimming ability of eels presented in our recent study can not exclude the possibility that recent declines in wild European eel populations may be due to eg. diminished natural food supplies. Also other factors like parasites (Haenen 1995), pollution, viruses (van Ginneken et al. 2004, 2005a) and restocking programs with weak slow growing animals from aquaculture can ultimately have its impact on the quality of the standing population.

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7) Migration and spawning depth There is only one published study looking at eel migration at great depth where a migrating eel with a swollen belly was photographed in the waters off the Bahamas at 2000 m depth (Robins et al. 1979). Changes in body characteristics like enlargement of the eyes (Pankhurst 1982, Pankhurst and Lythgoe 1983) and the silvering of the body during metamorphosis from yellow towards the silver stage (Tesch 2003) have been documented as physiological and morphological adaptations to a new life phase in the oceanic environment. The visual sensitivity of the retina pigments also changes from green-sensitive to blue-sensitive during metamorphosis of the European eel (Wood and Partridge 1993; Archer et al. 1995). Interestingly, indirect evidence suggests that migratory adults are adapted endocrinologically and physiologically for swimming and spawning within the upper 500 hundred meters, the epi- and upper meso-pelagic zones. Endocrinological evidence came from a field study by Dufour and Fontaine (1985) where cages with silver eels were sunken in the Mediterranean Sea at a depth of 450 m. Positive results which are indicative for maturation were recorded; a slight increase in ovarian development (GSI of 1.56 in control group compared to a GSI of 2.18 in pressure exposed group) was observed while the pituitary gonadotropin content increased by a factor 27 compared to the control group. Physiological evidence came from the observations of eel swimbladders and their ability to maintain swimbladder volume at depth. It is hypothesised that migration and spawning occur in the pelagic zone of the upper two hundred meters (Kleckner 1980). Swimming with a swollen belly due to gonad development might not be very energetically efficient from a hydrodynamic point of view. Therefore, during the first part of the migratory passage development of the gonad has to be delayed; swimming at depths with temperatures less than 10o C can postpone the maturation process. This assumption is based on the observation of artificial maturation experiments with hormonally treated eels showing that the development of the gonad is temperature dependent. Full sexual maturation in male Anguilla anguilla takes about 20 days at 25o C and about 60 days at 15o C, but gonadal development does not progress at temperatures below 10o C (Boetius and Boetius 1967, 1980). During initial migration low temperatures may be selected while upon arrival at the spawning area, eels have access to warm surface layers that accelerate maturation in preparation for spawning. There are several indications from fisheries harvest and telemetry studies, which provide direct evidence for eels swimming and spawning at relatively shallow depths. However, care has to be taken in the interpretation of these data because the number of eels caught or

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sampled is very low. Silver phase A. anguilla have been caught in the eastern North Atlantic by pelagic trawls towed at maximum depths of 325 m (Ernst 1977). A migrating maturing female eel has been caught at a depth of 500 meter close to the Azores in a deep sea trench of 2000 m. This eel had a GSI of 10 and gonads containing oocytes at advanced stage 3 (Bast and Klinkhardt 1988). Silver eels have also been recovered from stomachs of bottom- dwelling fishes captured at depths of more than 700 m (Reinsch 1968). Several telemetry studies gave information about depth and temperature preference in eels. Again care has to be taken in interpreting these data because the number of studies and animals is low. A second point of concern is the extrapolation of results from telemetry studies in relatively shallow coastal waters to the deep ocean. Silver eels, which were tracked when they left the continental slope off the Bay of Biscay and west of Spain, occupied depths of at least 400 m, but selected shallower depths (50 to 215 m) at night (Tesch 1978). Studies in the western Mediterranean Sea tracking eels provided information on thermal preference. Eels tended to swim in the 13o C hypolimnion, but regularly crossed the thermocline during vertical migrations (especially at night) into surface waters as warm as 18o C. Preferred depth at night was 196 m and 344 m during daylight (Tesch 1989). In a laboratory experiment, final preferred temperatures (FPT) of adult pre-migratory and migratory American eels were determined using chronic tests in a horizontal thermal gradient. The Final Preferred Temperature (FPT) is the temperature an animal ultimately selects in a horizontal thermal gradient after chronic exposure. Results indicated that both mature and non-developing Anguilla rostrata in saltwater had mean FPTs of 17.5o C, which is indicative of selection for a relatively high temperature (Haro 1991). Most observations from field studies indicate that migration of adult silver eels occurs in the upper 500 meters of the open ocean and is a shallow-water phenomenon. There is a clear diurnal rhythm with eels occupying shallow, warm depths at night and diving to deeper, colder depths during the day to avoid high light intensities (Tesch 1989). Information about the depth of spawning has been extrapolated from data on the release of hormone treated females tagged with transmitters, and on larvae catches. Again, the number of telemetry studies and the number of radio transmitter tagged animals is low. Releasing hormone treated mature female adults tagged with radio transmitters in the Sargasso Sea demonstrated a preference for the upper zone of the ocean at depths of 250-270 meters and at temperature 18.7-18.8 o C (Fricke and Kaese 1995). However, in the study of Tesch (1989) the maximum swimming depth of hormone treated silver female eels in the Sargasso Sea was nearly 700 m. Hormone treated female Japanese silver eels tagged with ultrasonic

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transmitters were released at their supposed spawning grounds in the western Pacific Ocean near sea-mounts on the West Mariana Ridge. These eels preferred relatively shallow water, swimming at a depth ranging from 81-172 m and at relatively high temperatures of 18-28o C (Aoyama et al. 1999). Interestingly, the catch of Anguilla larvae < 5 mm confirmed these observations. The smallest (probably just hatched) larvae were found at depths between 50 and 300 m with temperatures of 18o to 24o C respectively (Castonguay and McCleave 1987). Those temperatures are close to the final preferred temperature (FPT) of sexually mature Anguilla rostrata (17.5o C), so spawning probably takes plays in the upper 200 meter of the ocean at temperatures close to FPT (Haro 1991).

8) The spawning period

The major question regarding the timing of spawning is whether the putative spawning time derived from collections of small leptocephali is compatible with departure times and swimming estimates for silver eels. Usui (1991) reported that male European silver eels (approximately 40 cm) depart as early as August from the European coast to the Sargasso Sea, while female silver eels (mean body length ≥ 50 cm) depart one or two months later during September-October. It is possible that female eels arrive later in the coastal areas because they dominate low density up-river populations and thus have further to migrate downstream before reaching the sea. In contrast, males live in lower coastal areas and lagoons (Tesch 1977). Another explanation is that because of the males’ smaller body length they have a lower cruising speed. Assuming a cruising speed of 1 BL/sec, males would perform the 6000-km journey in 174 days, while females could perform the journey to the Sargasso Sea in only 139 days. The difference in migration time between males and females corresponds to approximately one month which could explain why males depart one month earlier. Ultimately males and females will meet each other in the Sargasso Sea to spawn as reported by Schmidt (1923) and the different publications produced by the group of McCleave and the group of Tesch (for references see section 2). Assuming a swimming speed between 0.5-1.0 body length per second they will reach the Sargasso Sea exactly six months later in the same period when recently hatched larvae have been observed: from March into June for the European eel and from February into April for the American eel (Kleckner et al. 1983; McCleave and Kleckner 1985; McCleave et al. 1987). So spawning of the European and American eel species is partially sympatric in space and time (McCleave et al. 1987) (see also section 2)

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The assumed swimming speed of 0.5-1.0 body length per second for endurance performance of eels was based on two types of laboratory experiments. Measurements of stress hormones, substrates, the ionic balance and lactic acid with groups of eels at different swimming velocities up to 3 BL/sec. From these experiments we conclude that a relatively low cruising speed of up to 2 BL/sec for eels could be characteristic for this catadromic long distance traveller (van Ginneken et al. 2002). The second type of experiment was based on oxygen consumption data which gave similar results (Palstra, Leiden University, The Netherlands, unpublished data).

9) Spawning behaviour and reproduction Although the literature on hormone-induced reproduction in eel species is extensive (see reviews: Ohta et al.1997, Pederson 2003, van Ginneken et al. 2005d, Palstra et al. 2005), no clear descriptions are given of spawning behavior of eels in the laboratory. Most aquaculture literature on this topic generally describe ‘stripping procedures’ to mix fertile eggs and sperm. The only report in literature that gives ethological data is a report by Boetius and Boetius (1980) which photographed a male eel in an aquarium releasing sperm with a mature swimming female with a swollen belly. In order to answer the question if spawning occurs at the surface, our laboratory made observations of spawning behavior with hormone treated animals. Three types of spawning behavior were documented during this experiment (van Ginneken et al. 2005d) female-female, male-female and male-male interactions (figure 6). The two females (1.5-2 kg) that were used in this experiment hung lethargically for hours or cruised together (33.6% and 66.4% respectively) (van Ginneken et al. 2005d). Male-female interactions we observed showed sperm release by several males with one female. In relative percentages, the different forms of spawning behavior can be classified as follows: (a) approaching the head region of the female (57.7%); (b) touching the operculum (39.4%); (c) approaching the urogenital area (2.9%) by the males (total observation 725 sec, figure 6 ). Non-sticky pelagic eggs were released to the surrounding water and the parents showed absence of parental care so their behavior can be classified as non-guarding (van Ginneken et al. 2005e). Maximum speed of eggs rising to the surface in a water column was 2.24 ± 0.33 metres (m) per hour (h). Male-male interactions involved both males chasing each other seldomly releasing sperm. (van Ginneken et al. 2005d). This study is an important observation documenting for the first time spawning behaviour in

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European eels. Based on these observations we concluded that induced spawning of European eel was collective and simultaneous possibly triggered by pheromones (van Ginneken et al. 2005d).

10) Maturation of European eel by environmental factors One of the mysteries of the life cycle of the European eel is the endocrinological mechanisms which induce maturation of the gonads during their catadromous migration to the Sargasso Sea. When eels first migrate to the ocean in autumn, there is limited development of the gonad (GSI. =1-2). If we keep these animals in aquarium boxes, there is no further development of the gonad; external environmental triggers for gonad maturation are lacking. Dufour (1994) demonstrated, a prepubertal neuroendocrine blockage in the European eel at in the silver stage. Gonadotropin-releasing hormone (GnRH) in the pituitary is deficient in this blockage, and an inhibition by dopamine was found (Dufour 1994). Both factors are responsible for the lack of production of gonadotropin (GTH) by the pituitary and a blockage in the release of GTH resulting in immature gonads.

Figure 6: Spawning behavior of artificially matured European eel (Anguilla anguilla L.). Two females were used, together with successively 3 trios of males to record their spawning behavior in the 4000-liter aquarium: A) Male stimulates female at the head region, B) Male attracted by the urogenital region of the female, C) Mass spawning, several males with one female with release of sperm, D) Interaction between females. Two females chasing each other. Induced spawning behavior of eels was massive and simultaneous. (van Ginneken et al. 2005d).

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This led to the hypothesis that sexual immaturity in silver eels is caused by a dual blockage situated in the hypothalamous-pituitary axis of the brain. The endocrinological mechanism by which this dual blockage is reversed is not yet clear. Although no adult eel has ever been caught in the Sargasso Sea to determine GSI, observations of hormone treated animals showed that GSI values in mature animals may vary between 40-70 (references in van Ginneken et al. 2005d). Based on these observations, we conclude that maturation and development of the gonad is triggered by external environmental factors that the animals are exposed to during their 6,000-km migration to the Sargasso Sea. It is not yet known which environmental factors can induce a final maturation of the animals.

For the maturation of migrating silver eel several environmental stimuli have been suggested, including temperature (Boëtius and Boëtius 1967), light (Nilsson et al. 1981), salinity (Nilsson et al. 1981) and pressure (Fontaine 1993). The latter factor is based on one observation of a migrating eel with a swollen belly at the Bahamas at 2,000-m depth (Robins et al. 1979).

The first three environmental factors (temperature, light, and salinity) have been found to have no clear effect on the hypothalamo-pituitary-gonad axis in eels (Boetius and Boetius 1967; Nilsson et al. 1981). Water pressure has been investigated in the laboratory (Sebert and Barthelemy 1985, Simon et al. 1988), as well as in field studies (Dufour and Fontaine 1985). Laboratory studies with eels placed under pressure at 2.5 Mpa (Nilsson et al. 1981) and 101 at- mospheres (Sebert and Barthelemy 1985; Simon et al. 1988), physiological changes were observed in the metabolism but not in maturation of the gonads. This was still the case after long term exposure to high-pressure of one month (Simon et al. 1988), or four months (Nilsson et al. 1981). Only one study has recorded a stimulation of the HPG-axis. In this field study (Dufour and Fontaine, 1985), cages with silver eels were sunk in the Mediterranean Sea at a depths of 450 m. This resulted in a slight ovarian development; the authors reported a GSI of 1.56 in the control group compared to a GSI of 2.18 in the pressure exposed group. But the most remarkable change was the observation that the pituitary gonadotropin content increased by a factor 27 compared to the control group (Dufour and Fontaine 1985). Remarkably, physical exercise has never previously been investigated as a potential stimulating factor. We hypothesise that maturation can be induced by exercise, because enormous physiological and endocrinological changes are the result of exercise in catadromous and anadromous fish species (Smith 1985). The Leiden group performed experiments to investigate

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the effect of long distance swimming on the HPG- axis and cortisol levels of European eel. Therefore, we studied the effects of swimming performance on maturation parameters in European eel (Anguilla anguilla L.) in large (127 l) Blazka swim-tunnels, (unpublished results). Three year old hatchery eels (71.4 ± 4.2 cm) with a mean weight of 792.0 ± 104.3 g were used in this study. One group of eels swam for 173 days at 0.5 body length per second and covered a distance of 5533 ± 354 km. One group was kept in static water for 173 days (Rest group). A control group was sampled at the start of the experiment in order to determine the initial stage of reproductive development. At the end of the swim trial, the maturation parameters 11-ketotestosterone, pituitary levels of LH and plasma levels of estradiol were higher (although not significantly) in the swim compared to the rest group. This observation can be explained by some animals responding and others not. In addition, no significant differences were observed in most measured morphometric and reproductive parameters, including eye-index, gonadosomatic index, hepatosomatic index, and plasma levels of vitellogenin, cortisol and melanophore-stimulating hormone (MSH). Also, pituitary levels of both MSH, and adrenocorticotropic hormone (ACTH) were unaffected. In contrast, the oocyte diameter was found to be significantly higher in the swim compared to the rest group. Based on these observations we conclude that a period of prolonged swimming might be a physiological stimulus necessary for the onset of maturation in the European eel (unpublished results).

11) Tracking silver eel migrations

To date no silver eels have been caught either on migration in the open ocean or in the Sargasso Sea. Schmidt’s hypothesis that Anguilla anguilla spawns in the western North Atlantic thus rests on the distribution of newly-hatched larvae in the Sargasso Sea, near the assumed centre of spawning (Tesch 1982; Schoth and Tesch 1982; Wippelhauser et al. 1985; Castonguay and McCleave 1987; McCleave and Kleckner 1987; Kleckner and McCleave 1988; Tesch and Wegner 1990). Conclusive evidence in favour of Schmidt’s hypothesis could be obtained in several ways, if the appropriate methodology were available. The location of spawning grounds could be deduced from the distribution of adult fish, if it were possible to catch silver eels in spawning condition at sea. The Sargasso Sea is, however, about 5000 m deep and limited fishing with mid-water trawls to a depth of 2000 m (Post & Tesch, 1982) has so far only succeeded in catching adult eels of the genus Serrivomer.

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Another approach would be to delineate the distribution of newly spawned eggs, but first it would be necessary to develop a method of differentiating the eggs and early larvae of A. anguilla from those of other anguillid eels. Because this cannot be done using morphological characteristics, a molecular approach would be needed (Watanabe et al., 2004). A third option would be to track the movements of silver eels throughout their migration from European rivers back to the Sargasso Sea using electronic tags.

Small archival tags (e.g. Metcalfe & Arnold, 1997; Arnold & Dewar, 2001) are eminently suitable for tracking individual fish over periods of a year or more. Temperature and pressure sensors provide information about the vertical movements of the fish in relation to the thermal structure of the water column and horizontal movements can be reconstructed using light-based geolocation techniques (e.g. Hill, 1994; Arnold & Dewar, 2001; Ekstrom, 2004; Stokesbury et al., 2004; Teo et al., 2004). However, although useful for small demersal fish such as plaice and cod (e.g. Metcalfe & Arnold, 1997) and large pelagic fish such as bluefin tuna (e.g. Block et al., 2005), archival tags are of little or no use for species, such as eels, where recapture rates or low or non-existent. For these species, the only practical alternative is to use pop-up archival tags (Block et al., 1998) programmed to detach themselves from the fish on a specific date, float to the surface and transmit archived data by radio to Service Argos (e.g. Taillade, 1992). This service, which is based on a series of polar orbiting satellites established by the National Oceanographic & Atmospheric Administration (NOAA) in the USA and operated by CLS in France (http://www.cls.fr), offers a commercial service for remote data retrieval with two frequencies (401.648 & 401.652 MHz) dedicated to animal telemetry.

Pop-up archival tags, which are currently made by two commercial companies (Microwave Telemetry, Columbia, Maryland; Wildlife Computers, Redland, Washington) in the USA, have been used successfully on various species of sharks (e.g. Sims et al., 2003; Boustany et al., 2002), as well as tuna (e.g. Lutcavage et al., 1999; Block et al., 2001; Block et al. 2005) and billfish (Holland, 2003). They consist of an archival tag with light, temperature & pressure sensors, a radio transmitter and a microprocessor, which controls the release mechanism, as well as data recording and processing. Electronic circuits, sensors and batteries are contained within a cylindrical case (approx. 110 x 20 mm) with a nose-cone at the front and a large polystyrene float (approx. 55 mm long x 40 mm diameter) at the rear. The nose-cone contains the mechanical components of the electrolytic release mechanism.

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A quarter wave length radio antenna (216 mm long) protrudes from the end of the float. The tag is towed horizontally behind the fish, but floats vertically after it has been detached. Surface transmission time typically varies between 10 and 20 days.

Although some preliminary studies have been carried out in New Zealand with female long- finned eels (Anguilla dieffenbachi) of 7.6-11.4 kg weight (Jellyman & Tsukamoto, 2002), it is not practical to use pop-up tags with European eels unless the size of the tags can be reduced substantially. Whilst miniaturisation of electronic circuits and sensors and a reduction in battery size would help considerably, the main challenge is to develop a smaller flotation mechanism that imposes less drag on the swimming fish, but at the same time allows more effective radio transmission. This may be possible by replacing the solid float with a device that inflates after the tag is detached and is capable of lifting the radio aerial clear of the water after the tag has reached the sea surface. Pressure resistance must be increased to allow tags to operate at depths beyond the current limit of 2000 m and research is also needed into tag attachment methods to avoid the problem of premature release that commonly occurs in bluefin tuna (e.g. Wilson et al., 2005) and a number of other species.

If these difficulties can be overcome, small pop-up archival tags could provide the key to discovering the routes that silver eels follow during their spawning migrations. Although existing light-based geolocation techniques (Hill, 1994; Arnold & Dewar, 2001; Musyl et al., 2001; Ekstrom, 2004; Stokesbury et al., 2004; Teo et al., 2004) are sufficient to describe overall patterns of oceanic movement, more accurate estimates of position may become possible by including a hydrophone to receive sonar signals transmitted by a buoy or a ship. One approach might be to record location each time the fish passes within a few kilometres of a ship or a buoy, whose position is determined by GPS and transmitted underwater via an encoded sonar signal (Gudbjornsson et al., 2004). Technology to do this in the open sea, in areas covered by regular research vessel surveys, has been developed by Star-Oddi (Reykjavik) and is under evaluation in one of Iceland’s largest fjords (www.star-oddi.com). Another, and possibly more suitable technique, would be to use a miniature version of the RAFOS float system and determine position by triangulation, using a miniature processor in the tag to compare the reception times of signals from three or four moored transmitters (Lee et al., 2002). This system is currently under development.

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Although we presented a recent update of the literature on the life cycle, evolution and reproduction of eels in this review, still some major questions remain to be answered: 1. Where are the exact spawning grounds for different eel species? 2. What is the environmental stimulus for reproduction and does it vary by species? 3. What are the critical habitat and environmental parameters needed for successful reproduction for the eel in the wild? 4. What are the causes of the decline in the eel population and their genetic consequences on the long term ?

Generation after generation, scientists have dedicated their time and energy to study the catadromous European eel (Anguilla anguilla L.). Although a long way has been covered since Aristotle’s theory of spontaneous generation in eels, the endless quest to unveil the fascinating life cycle of this mysterious creature will ultimately have to take us back to the Sargasso Sea, where everything started. For 3 million years this species succeeded in maintaining its characteristic life style with a remote spawning in the tropical North Atlantic Ocean and a juvenile foraging life phase till partial maturation in freshwater systems on the European continent. However, the last two decades eel populations declined dramatically by 90-99%, probably due to the synergy between human activities and oceanic fluctuations, bringing this species to the brink of extinction. Not much time remains to pinpoint the real causes of this decline and consequently to prevent the irreversible loss of this mysterious species.

Acknowledgments Vincent van Ginneken was supported by a grant of the Technology Foundation (STW), which is subsidised by the Netherlands Organisation for Scientific Research (NWO), STW- project no. LBI66.4199 and by the European Commision (Project QLRT-2000-01836). G.E. Maes was funded by a PhD fellowship from the I.W.T. (Institute for the Promotion of Innovation by Science and Technology in Flanders and is currently funded by a post-doctoral fellowship at the Catholic University Leuven . Dr. Geoff Arnold is very much acknowledged for his contribution in writing the telemetry paragraph (section 11). The editor of Reviews in Fish and Fisheries Dr. Jennifer L. Nielsen and Prof. Dr. M. Richardson (Leiden University) are acknowledged for critically reading the manuscript and helpful suggestions for correction of the English.

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278 Annex 2

Gonad development and spawning behavior of artificially-matured european eel (Anguilla anguilla L.)

Vincent van Ginneken1, Gerjanne Vianen1, Bie Muusze1, Arjan Palstra1, Louise Verschoor1, Olivier Lugten1, Marjolijn Onderwater1, Sjoerd van Schie1, Patrick Niemantsverdriet1, Richard van Heeswijk1, Ep Eding2, Guido van den Thillart1

1. Department of Integrative Zoology, Institute of Biology Leiden, van der Klaauw Laboratories, P.O.Box 9511, 2300 RA Leiden, The Netherlands. 2. Department of Fish Culture and Fisheries, Wageningen Agricultural University, Marijkeweg 40, P.O.Box 338, 6700 AH Wageningen, The Netherlands.

Corresponding Author: Dr.Ir.V.J.T.van Ginneken, Department of Integrative Zoology, Institute of Biology Leiden, van der Klaauw Laboratories, P.O.Box 9511, 2300 RA Leiden, The Netherlands, FAX: +31(0)71-5274900, E- mail: [email protected] , TEL: +31(0)71-5277492

Published in : Animal Biology, vol. 55, No 3., pp 203-218 (2005)

279

Annex 2

Geslachtsdifferentiatie van de paling ’Aelen en sijn wijfkens noch mannekes ende sij hebben gheen eyeren maar si worden gheboort uten slijm ende vuylich der ander vissche’ (Coenenz.A.; Visboeck 16e eeuw, MS Koninklijke Bibliotheek, Den Haag, 78E54).

Annex 2

GONAD DEVELOPMENT AND SPAWNING BEHAVIOR OF ARTIFICIALLY- MATURED EUROPEAN EEL (ANGUILLA ANGUILLA L.)

ABSTRACT

Gonadal development and spawning behavior of artificially-matured European eel (Anguilla anguilla L.) was studied. Treatment of males with Human Chorionic Gonadotropin (HCG; 1 IU gr per week) resulted in a GSI of 10.88 ± 3.39 and spermiation. Treatment of females with carp pituitary suspenion (20 mg cPs per kg body weight per week) resulted in oogenesis with a GSI of 20.0 ± 11.3 (n=7), and the number of eggs per female was 1874 *103 ± 1116 *103; (n=7). Ovulation of the females was induced with 17α, 20β dihydroxyprogesteron (DHP) at 2 μg per gram bodyweight. Eggs of European eel are found not sticky and typically pelagic. Maximum speed of eggs rising to the surface in a water colomn was 2,24 ± 0.33 meters (m) per hour (h). To study behavior in a qualitative way, two females were used together with three groups of three males. During a 283 minute (min) observation of the two females, we observed female-female interaction: 'lethargic behavior' (33.6%) versus 'together cruising' (66.4%). In the period when males and females were together (188 minutes), we observed 'approaching the head region of the female' (57.7%), 'touching the operculum' (39.4%), or 'approaching the urogenital area' (2.9%) by the males (total 725 seconds (s)). Sperm release in the presence of a female took 115 s of the total approaching time of 725 s (15.9%), while in the case of male-male interaction this was only 15 s of the total period of 116 s (12.9%). Induced spawning behavior of eels was collective and simultaneous, corresponding to spawning in a group. This is the first time spawning behavior has ever been observed and recorded in eels.

INTRODUCTION

When eels migrate to the ocean in the autumn, their gonads are regressed. If they are kept in aquaria, there is no further gonadal development. Based on these observations, it seems that maturation can be triggered by environmental factors during migration. Until now, the nature of these factors was unknown.

280 Annex 2

Some of these possible external factors are: (i) hydrostatic pressure (Nilsson et al. 1981; Fontaine et al. 1984; Dufour & Fontaine 1985; Sebert & Barthelemy 1985; Simon et al. 1988), (ii) low temperature (Nilsson et al. 1981), or (iii) swim exercise (Palstra et al., unpubl.; van Ginneken et al. unpubl.). It has recently been demonstrated, in European eels in the silver stage, that there is a prepubertal blockage of gonadal maturation at the neuroendocrine level because of a deficiency of gonadotropin-releasing hormone (GnRH) at the level of the pituitary, resulting in a lack of production of gonadotropin (GTH). Furthermore, there is an inhibition of GTH release by dopamine (Dufour 1994). Both factors are responsible for an immature gonad. The main obstacle to successful aquaculture of European eels is the production of viable eggs and larvae. For reproduction of most fish species, only the final part of the gametogenesis, final oocyte maturation and ovulation, has to be induced. In contrast, the entire reproduction cycle, including endogenous and exogenous vitellogenesis and also ovulation, has to be induced in eels. Several authors applied the protocol for artificial administration of hormones. If we consider the research on this topic chronologically, much attention has been paid to the production of gametes and the production of viable larvae, while little study was carried out on behavioral aspects of mating eels. As early as 1936, Fontaine had obtained sperm from male eels, which were treated with Human Chorionic Gonadotropin (HCG; Fontaine 1936). Several other authors also performed artificial stimulation of sperm production (Boetius & Boetius 1967; Dollerup & Graver 1985; Miura et al. 1991). Yamamoto & Yamauchi were the first who obtained eel larvae from Anguilla japonica (Yamamoto & Yamauchi 1974; Yamauchi et al. 1976). Boetius & Boetius (1980) injected A. anguilla with pituitary extracts and published a photograph of a spermiating male close to a swimming mature female. Bezdenezhnykh and Prokhorchik described embryonic and post-embryonic development of European eel larvae until 3.5 days post-hatch. However, details about the methods were not published in their papers (Bezdenezhnykh et al. 1983, Prokhorchik 1986, Prokhorchik et al. 1987). Ohta et al. (1996) showed that the addition of Dihydroxyprogesterone (DHP) is crucial for the induction of ovulation. Lokman & Young (2000) applied the protocol of Ohta to the New-Zealand eel (A. dieffenbachi, 5 kg) and demonstrated that DHP also induced ovulation in this species. Pedersen et al. (2003) applied the procedures of Ohta et al. (1996) to Japanese eel (A. japonica) and, using the same procedures, also succeeded in producing larvae from European eel (A. anguilla) (Pedersen, 2003, 2004). Recently, our group was able to fertilise egg batches of nine females and

281 Annex 2 development of 1600 embryos was followed until 4 days after fertilisation (Palstra et al., in press). Hatching had not been observed. From the above studies, it is clear that induced sexual maturation of European, Japanese, and New Zealand eels may lead to fertilized eggs and viable larvae. However, when the procedure of 'stripping' (removing sexual products from the animals by gentle pressure to the abdomen) is applied, natural behavior does not occur and spawning behavior cannot be studied. Therefore, we hypothesize that if artificially matured eels are put together in an aquarium, spawning behavior can be observed. Until now, the spawning behavior of eels has never been described, because the spawning grounds of the various eel species are in the Sargasso Sea, several thousand kilometers away from land. Our laboratory approach will give new insights into the life-cycle and the mating behavior of this catadromic fish species.

MATERIAL AND METHODS

Hormones and Chemicals To induce oogenesis in females, carp pituitaries were obtained from a commercial company "Catfish", (Den Bosch, The Netherlands). Ovulation was induced in females with 4- pregnene-17α, 20β-diol-3-one (17α, 20β dihydroxyprogesteron (DHP) (Sigma Aldrich Chemie BV, Zwijndrecht, The Netherlands). To induce spermiation in males, Human Chorionic Gonadotropin (HCG) was used (Sigma Aldrich Chemie BV).

Preparation of the hormone solutions For the preparation of the carp pituitary extract, portions of 0.5 g carp pituitaries were weighed and each portion was ground with a pestle in 5 ml saline solution (9 %o NaCl). The suspensions were supplemented with 10 ml buffered saline and homogenized with an ultratorax (2 x 5 seconds at 3000 r.p.m.). Next, the 'carp pituitary suspension' (cPs), was treated for 10 minutes in an ultrasound bath (2-4 °C). After 12 h storage on ice, the suspension was centrifuged for 10 minutes at 20000 g, 4°C. Next, the supernatant was pipetted, recentrifuged and stored at -80°C in 1 ml aliquots. The final ratio of carp pituitary vs. buffered saline solution was 1 g of carp pituitary suspended in 40 ml saline solution. For the preparation of the ovulation hormone, 10 mg of DHP was dissolved in 875 μl 100% ethanol. Of this solution, 175 μl per kg was diluted with saline solution (1:1) and injected intraperitoneal at several locations in the ovary.

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Growth of the ovary with cPs and the induction of ovulation Nineteen females (range 0.7-2.5 kg), each identified with microchips (Trovan), were kept in a recirculation system of 1000 liters in artificial seawater (33 promille) at a temperature of 18°C. The eels were caught in the autumn of 2000 in lake Grevelingen, The Netherlands, during their seaward migration. The maturation experiments were performed in the period April-August, 2001. Ovarian growth of each animal was stimulated by weekly IP injections of 20 mg cPs per kg body weight. On the day of the injection, and to prevent bacterial infections, the animals were exposed to the antibiotic Flumequine (50mg/L) for 3 h in a separate tank. Body weight was measured. After 8 weeks (8 injections), body weight was measured 2 days after the injection. Two days after cPs injection, body weight increased by more than 10 %. At this point, a sample of the eggs was taken with a cannula and investigated under a binocular-microscope. If the eggs were opaque, the female was not ready to ovulate. When most eggs had undergone final oocyte maturation, signified by their becoming transparant, the female was selected for the ovulation protocol. After 14 weeks of treatment, the first females were ready for the induction of final oocyte maturation. Most females had mature oocytes after 14-25 injections of HCG. When a female was selected after injection with dihydroxyprogesterone (DHP) together with a ‘booster’ injection of cPs (20 mg cPS per kg body weight), she was placed in a 1500 liter aquarium at a temperature of 20°C (temperature shock of 2° C). Ovulation could be observed after 16-24 hours by gently pressing on the belly of the female. In fertilization experiments, ovulated eggs were stripped without force. Of the nineteen females used in the experiment, seven females were finally selected for ovulation, while two females were used to film spawning behavior. Ten females died during the injection protocol. In order to avoid handling stress during the whole injection protocol over the period of 14 weeks, animals were on the day of injection anaesthetized in a soltion of 3-aminobenzoate ethyl ester methanesulphonate (MS-222,

Sigma, St.Louis, MO) buffered with NaHCO3 at a final concentration of 200 ppm.

Sperm production In the autumn of 2000 approximately 100 male eels (100-150 g body weight) were caught by a local fisherman at the Grevelingen-lake during the eels’ seaward migration. Weekly injection of the males started in April 2001. In order to exclude pheromone effects, they were kept separate from the females in a re-circulating system of 1500 liters in artificial

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seawater (33 promille). Concerning water quality, the maximum values for NH4, NO2, and

NO3 were 0, 0, and 50 mg, respectively, per liter, at 18 °C. Growth of the testis of each animal was induced by weekly IP injections with Human Chorionic Gonadotropin (HCG) at a dosage of 1 IU per g body weight per week (Ohta et al. 1997). The first males released sperm after 6 weeks. Sperm motility in seawater was checked under a microscope. Also, body weights of males were recorded every week.

Speeds of eggs rising to the surface In six cases, released water-activated eggs rising in the water column were quickly transferred to a 2000ml glass beaker. Each individual egg was released at the 500ml bar and the required time for the egg to rise up to the 1000ml bar (= 6.4cm) was measured. From these data speeds were calculated and averages ± standard deviation were expressed in meters per hour and meters per day.

Experimental set-up for spawning behavior Two chronological protocols were used to observe and study spawning behavior. To achieve this, the eels were allowed to swim freely in a 4000-liter aquarium. In the control protocol, two females were used. To study spawning behavior, the same two females, followed by 3 sequential batches each of three males, were used. Our observation period of only female- female interaction lasted 283 minutes. The period of female-male and male-male interaction lasted 188 minutes. The first batch of three males was for a period of 83 minutes with the two females, the second batch of three males for a period of 50 minutes, and the third set of three males for a period of 55 minutes. We used indirect light so as not to disturb the eels. We could follow the experiment on monitors outside the laboratory. A Sony camera was used to record the spawning behavior of the eels on film.

Calculations and statistics: Statistics were performed using a one-way ANOVA. P≤ 0.05 was considered as statistically significant. Normality of the data and homogeneity of variances were checked by

Kolmogorov-Smirnov and Fmax tests, respectively.

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RESULTS

At the start of the experiment in April 2001, a control group of males was sampled and several zootechnical parameters were measured. The first HCG-treated males gave sperm as early as 6 weeks. After 12 weeks, 75 males gave 1-1.5 ml sperm per animal per week. The other 25 males did not respond to the hormone treatment and gave no sperm. In August 2001 after the maturation experiments were finished, 18 males, which were treated with hormones, were still alive: mortaility was high due to long-term hormonal treatment. The GSI of these animals was measured (see table 1). Figure 1 shows three mature males with developed gonads. At the start of the experiment in April 2001, a control group of females was sampled and several zootechnical parameters were measured before treating the females with cPs. No placebo-treated animals were studied because the injection with saline had no effect on gonad development for males (Boetius and Boetius, 1967) or females (Boetius and Boetius, 1980). Of the initial 19 females, six died during the first 14 injections while four others died at a later stage due to unknown causes. Of the 11 silver eels, which received at least 16 injections, seven matured and were treated with DHP. When the increase in bodyweight exceeded 10% of the original weight (Ohta et al., 1996), the animal was selected to induce ovulation via DHP injection. Seven females were stripped and zootechnical parameters of seven stripped females are presented in Table 2. Apart from GSI, the number of eggs was also determined. Figure 2 shows a mature female with a GSI of 40.2. Nuclear migration in an eel oocyte is illustrated in figure 3 (Germinal Vesicle Migration; GVM). Other characters of a ripe female were the swollen soft belly and a strong penetrating odor. Eggs rose to the surface at a speed of 1.58 ± 0.78 mh-1(one female: six trials with stripped and fertilised egs); however, individual differences were still high. The three fastest eggs rose at a speed of 2.24 ± 0.33 mh-1 or 53.7 ± 8.0 mday-1. Eggs were free rising to the surface and did not stick to the sides or to each other. Three types of spawning behavior were observed (figure 4):

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A: Control (n=12) B: HCG-treated C: HCG-treated P -value P -value P -value Positive responders Negative responders (N=11) (N=7) A-B B-C A-C Bodyweight (g) 132.94 (45.02) 82.55 (18.42) 72.50 (11.76) 0.002* 0.249 0.004**

Length (cm) 39.38 (1.79) 38.23 (2.33) 38.50 (2.59) 0.059 0.827 0.187

Gonad weight (g) 0.27 (0.26) 9.41 (4.68) 0.47 (0.27) 0.0001** 0.0001** 0.150

GSI 0.18 (0.116) 10.88 (3.79) 0.63 (0.34) 0.0001** 0.0001** 0.0001**

Table 1: Zoological parameters of Control- and HCG-treated males. Of the latter group eleven animals reacted positively on the hormone treated with HCG and produced sperm while seven HCG-treated males reacted negatively on the hormone treatment. P-values are given comparing groups: A-B, B-C and A-C. * denotes significantly different (P≤0.05) from Control group, ** denotes significantly different (P≤0.001) from Control group

Figure 1: HCG- treated mature males with a mean gonadosomatix index (G.S.I.) of 10.9 ± 3.79.

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Bodyweig Length Gonad GSI Weight egg Number of ht (cm) weight (mg) eggs x 1000 (g) (g)

Control - 1091.8 78.4 17.02 1.54 females (132.2) (4.37) (4.76) (0.30) XXX XXX Mean ± (SD) cPs-treated females 1 884 70 245 27.71 0.121 2,026 2 1664 85.5 399 23.98 0.101 3,945 3 656 71 63 9.60 0.067 950 4 744 71.5 73 9.81 0.068 1,066 5 746 69 96 12.87 0.124 772 6 1701 87 683 40.15 0.269 2,532 7 872 73.5 139 15.94 0.076 1,830 Mean ± (SD) 1038.14 75.36 242.57 20.01 0.118 1,874 (447.28) (7.581) (227.91) (11.26) (0.071) (1,115)

P-value Control vs. P≤ 0.907 P ≤ 0.650 P ≤ 0.001** P ≤ 0.001** cPs

Table 2: Gonad maturation parameters of a Control-female group of the European eel (Anguilla anguilla L.) and of a cPS-treated group.* denotes significantly different (P≤0.05) from Control group, ** denotes significantly different (P≤0.001) from Control group

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A: FIRST 1 hour and B: 50 minutes C: THIRD 55 minutes SET 23 minutes SECOND SET MALES SET MALES MALES Operculum Head Urogenital Operculum Head Urogenital Operculum Head Urogenital 4 31 3 6 13 0 30 9 3 9 40 3 6 11 0 32 3 16 60 3 10 114 0 33 25 3 13 28 0 115 3 4 37 0 8 18 0 28 0 24 0 80 0 MEAN ± MEAN ± MEAN ± MEAN ± MEAN ± MEAN ± MEAN ± SD MEAN ± MEAN SD* SD SD SD SD SD SD ±SD 9.67 ± 6.03 39.0± 3.0 ± 0 7.83 ± 3.25 28.11± 0 52.5±41.68 9 3.0 ± 0 15.30 21.24

Visit 8.67 Number of 18.0 Number of 7.64 frequency ** visits per visits per hour hour Duration of 144.58 Time of 360.0 Time of visit 245.45 visits *** visit in in seconds seconds per hour per hour

Table 3: Female seeking behavior of males. Mature males exposed to ovulating females approached these at females three different regions: the operculum, the head region or the urogenital area. Data from three consecutive trials (ABC), each with three males and two ovulating females. • Individual visit duration and mean duration of each type of visit, in seconds.; ** For each trial, the visit frequency (mean number of visits per hour) to the three regions; *** For each trial. The total duration of visits (in seconds per hour) to the three regions.

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Figure 2: Mature female of European eel with GSI of 40.2.

Figure 3: Oocyte showing Germinal Vescicle Migration (scale bar = 100μm). (phase contrast microscopy)

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Figure 4: Spawning behavior of artificially matured European eel (Anguilla anguilla L.) was studied. Two females were used, together with successively three groups of three males to record their spawning behavior in the 4000-liter aquarium. A) Male touching female at gill area, B) Male stimulates female at the head region, C) The release of sperm by two males with one female, D) Female-female interaction, females chase each other up.

Figure 5: Female seeking behavior of males. Mature males exposed to two ovulating females approached females at 3 different regions: the operculum, the head region or the urogenital area. The mean length in seconds of each type of behavior is shown in 3 consecutive trials with 3 males each (A,B,C).

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Female-Female Interactions:

During a period of 283 minutes, female-female interactions were observed (fig. 4 D). For the first 5 min, both females actively swam in the aquarium. Thereafter, a period followed during which both females hung lethargicly at the water surface. During periods of activity (totalling 95 minutes), one female approached the other towards the head region. Both females stayed together and swam together head-to-head for a certain period of time near the surface of the water (fig. 4 D). The relative percentage of 'lethargic behavior' vs. 'cruising together' was respectively 33.6% and 66.4%.

Male-Female Interactions:

The individual observations of behavior, split up for the three consecutive groups of three males (repetitions), are given in table 3 and figure 5. An hourly average was taken of the number of male visits made to different regions (operculum, head and urogenital) of the females. This was 8.7, 18.0, and 7.64 number of visits/h for male-groups A, B, and C, respectively. The total time spent visiting the different body regions of the female (operculum, head, urogenital region) was 144.6, 360 and 245.45 s/h for male-group 1, 2 and 3, respectively (Table 3). We observed sperm release by several males with one female (fig. 4 C). After an initial orientation phase of several minutes, males swam vigorously around pursuing a female, swimming parallel to her, and pushing her (fig. 4 A). Usually, the male approached the head region of the female and chased her for a short period of time, constantly touching and butting the operculum of the female with his head (fig. 4 A). After this, the male took on an S-like body shape, while ejaculating sperm near the urogenital region of the female (fig. 4 C), and swam away. The males repeated the chasing and the sperm ejaculation several times and released sperm at all regions of the female (head, operculum, urogenital region). On a few occasions, the male pushed at the urogenital region of the female with his head followed by the sperm release. Sometimes males ejaculated sperm, assuming an S-like shape. In these cases, they swam parallel to a female, with or without briefly butting her near the urogenital region. However, in most cases, the male did not assume this S-like body shape, but instead ejaculated his sperm as he passed by. On some occasions, a male chased a female while touching her operculum, after which the male swam away again, not releasing any sperm. In

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relative percentages, the different forms of behavior can be classified as follows: (i) approaching the head region of the female (57.7%); (ii) touching the operculum (39.4%); (iii) or approaching the urogenital area (2.9%) by the males (fig. 5). Occasionally, interaction of two males with one female was observed (fig. 4 A). Both males chased the female by touching her operculum while ejaculating sperm. The females also became active due to the spawning activities of the males. During the total observation period of 188 min, males approached the female several times, covering a period of 725 s. During this period, males ejaculated sperm during 115 seconds, which corresponded to 15.9% of the male-female approaching time. Sperm ejaculation occurred with all three sets of males. In the second set of three males, eggs were visible at the urogenital region of both females. With the last set of three males, one female released eggs. The males released so much sperm that no clear film images could be made due to turbidity. To overcome this problem, we put a proteinskimmer on our filter system in order to clear the water. Males were also attracted to the eggs on the floor of the aquarium, and to the urogenital tract of the female when the female released eggs. When the floor was totally covered with eggs, they were no longer able to find the females who stayed at the water surface.

Male-male interactions:

Males chased each other, only releasing sperm in a few cases. Sperm release lasted only 15 seconds (12.9%) of the total period of 116 seconds of male-male interaction. During ejaculation, males showed the same body movement (S-shape) as during sperm release in a male-female interaction.

DISCUSSION

Spawning behavior and gonadal development of artificially-matured European eel (Anguilla anguila L.) was studied in a 4000-liter aquarium. During the period of female-female interaction we distinguished: 'lethargic behavior' versus 'cruising together’. In the period of combined male and female interactions we observed 'approaching the head region of the female', 'touching the operculum', or 'approaching the urogenital area' by the males. Males reacted immediately after transfer.

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In this study on Anguilla anguilla, female GSI values ranged between 9.6 and 40.2; this maximal value is lower that the 60.7 reported by Boetius & Boetius (1980) for the same species. The highest GSI values recorded for female Japanese eels, A. japonica, treated with salmon pituitary extract, were around 70 (Yamamoto & Yamauchi, 1974; Sugimoto et al. 1976). The highest GSI reported for female American eels, A. rostrata, treated with cPs, was 44.8 (Edel, 1975). No GSIs were given in the literature for A. australis and A. dieffenbachi (Lokman & Young 2000). It is important to indicate if the GSI was determined before or after ovulation. If it is determined after Germinal Vesicle Migration (GVM) and Germinal Vesicle Breakdown (GVBD), the volume of the ovary will be much higher due to the uptake of water by the oocytes. Based on oocyte weight and GSI, we calculated that there were in the range of 772 000 – 3945 000 eggs per female. Dekker (2000) calculated, based on fisheries assessment data for the European eel stock, a recruitment of 2000 million glass eels. However, based on our fecundity data, an estimation of the total number of spawners needed in the Sargasso Sea to maintain the standing stock at the European continent is difficult to give because data on larval mortality during hatching in the Sargasso Sea followed by migration are lacking. It is more than likely that only a small fraction of the total number of eggs produced become reproductive adults that migrate back to the Sargasso Sea. In our study, the first males released sperm after 6 weeks of treatment with HCG. The rate of maturation is probably dose-dependent, which is confirmed by data in the literature. Male A. anguilla, given a dose of HCG twice as high as in our protocol, became mature after 8 weeks (Eckstein et al. 1982). Probably, the maturation rate is also species dependent because A. japonica males, given the same dosage as in our experiment, became mature after 9-12 weeks (Ohta et al. 1996). It is also possible that within a certain eel species temporal aspects and the maturation status of the animal might also be important and vary throughout the year. For the European eel the animals with the most progessive stage of development of gonads are found in fall (unpublished results) and that is why we collected our animals in this period prior to their seaward migration. So, maturation rate may not only be species dependent, but also reflect slightly different times during the annual cycle in which the hormones were administered. The small and numerous eggs of European eel were found non-sticky and typically pelagic. Therefore it is assumed that eggs will not stick to seaweed like Sargassum, typical for the Sargasso Sea. Pronounced hydration of eggs was observed in this study; this is typically seen in marine teleosts spawning pelagic (or buoyant) eggs (Wallace and Selman,

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1981). The acquisition of buoyancy in pelagic spawners is a key event in reproduction and effects both fertility and survival of spawned eggs (Carnevali et al., 1999). In this study, eggs were found to rise in the water column up to the surface with maximum speeds of 2.24 meters per hour. Main hatching times for European eel are between 47 and 60 hours after fertilisation (Bezdenezhnykh et al., 1983; Pedersen, 2003, 2004; Palstra et al., 2004a,b). During these times eggs will rise 105-134 m. Assuming that hatching occurs in the food rich upper water layers these shallow depths represent spawning depths. However, for Japanese eel it was observed that high pressure delays embryonic development and hatching times (Hiroi et al., 2003). Little is known about the Sargasso Sea, an enormous potential spawning area of 2 x 106 km2. At this moment, no information is available from the natural situation considering effects of population density, predation, light conditions, number of spawners etc. In a review, van Ginneken (unpublished results), summarized what is known about an estimation of the number of eel spawners, observations made on hormone-treated females in the Sargasso Sea which were followed by telemetry tracking, and the catch of adult spawners on their way to the Sargasso Sea. We are acutely aware that the present study, in a 4000 l aquarium, with two females and three groups of three males, has strong limitations in replicating the natural situation. However, this study is valuable because it is the first time that spawning behavior of eels has ever been observed and described. In the Sargasso Sea, sexual activity is probably limited to a restricted period. Therefore, the location of suitable mates, courtship and spawning must take place quite rapidly. It is likely that spawning is thus synchronised throughout the population. Information on the spawning behavior of European eel has to our knowledge never been reported before. The 'strong penetrating odor', excreted by a ripe female may contain a pheromone that induces spawning behavior. Natural spawning may be stimulated due to the action of pheromones (Colombo et al. 1982) which may induce ovulation and have a positive effect on hatching and fertilization rates due to a synchrony of gamete emission after massive and collective spawning of eels. Females were lethargic and waiting in the upper part of the aquarium to spawn. After an initial orientation phase, males swam periodically close to the females to court them. Also, we observed, for European eel, that spawning behaviour started immediately after transfer and was synchronous, showing several forms of social interactions: male-male, male-female and female-female. For the male-female interaction, spawning of one female with several males occurred occasionally. Several forms of behavior were observed including: i) lethargic behaviour: low activity as the consequence of the absence of a partner, ii) chasing behavior:

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which includes chasing or attacking of another fish without reciprocation, iii) submissive behavior: which we defined as the fleeing from an aggressive opponent, and iv) cruising: swimming over an area without being chased or followed. In the initial phase of the experiment, before males were introduced in the aquarium, females were hanging lethargical at the surface of the aquarium. After the introduction of males, the females became more active. The males mainly demonstrated aggressive behaviour. For male-male interactions there were no observations of males defending a specific territory, or specific dominance among males, or of males maintaining space around them. In a few cases during male-male interaction, when males chased each other, this behavior was followed by the release of sperm as was observed in male-female interaction. During male- female interaction, females demonstrated mainly submissive behavior. Non-adhesive, pelagic eggs were released to the surrounding water and the parents showed absence of parental care. Therefore their behavior can be classified as non-guarding. During our experiment, spawning took place in full light, and males were attracted to the eggs (without eating them) probably by odour. Therefore, crepuscular spawning which takes place with other fish species to prevent predation on zygotes (Roberts & Ormond 1992) is probably not important for the European eel. During female-female interaction we observed mainly cruising behavior. Both females stimulated each other by swimming close to each other and touching each other. This behavior may be important to induce a synchrony of gamete emmision and induce a massive simultaneous spawning of numerous mating eels in the Sargasso Sea.

ACKNOWLEDGMENTS

We thank Dr. Lex Raat and Drs. Jan Klein Breteler, Organization for Improvement of Inland Fisheries, Nieuwegein, Netherlands, for supporting this project. Dr. Ir. Marc Lokman, Zoology Department, University of Otago, Dunedin, New-Zealand is kindly acknowledged for his help and advice. Dr.Ingo Schlupp, University of Hamburg, Germany, for helpful advice and fruitful discussions on ethological issues. Dr. Herman Berkhoud and Jos Onderwater for the beautiful pictures. Furthermore, the authors wish to thank Edwin Cohen and Eugenia Clavero for assistance. We thank Prof. Dr. M. Richardson for critically reading the manuscript and helpful suggestions for improving of English. The eel migration project at the University Leiden is supported by a grant of the Technology Foundation (STW), which is subsidized by the Netherlands Organization for Scientific Research (NWO), STW-project no. LBI66.4199.

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Literature

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Fontaine, M. (1936). Sur la maturation complète des organes génitaux de l'anguille male et l'émission spontanée. C.r.hebd.Séanc.Acad.Sci.Paris 202, 132-1314. Fontaine, Y-A., Dufour, S. & Fontaine, M. (1984). Un vieux problème très actuel: la reproduction des anguilles. La Vie des sciences, Comptes rendus 2, 1-10. Hiroi, J., Yasumasu, S., Kawazu, K., & Kaneko, T., 2003. Hatching enzymes in the Japanese eel. In: Aida, K., Tsukamoto, K., Yamauchi, K., (Eds.), Eel Biology pp.445-456, Springer Verlag, Tokyo, Japan. Lokman, P.M. & Young, G.( 2000). Induced spawning and early ontogeny of New Zealand freshwater eels (Anguilla spp.) . New Zealand Journal of Marine and Freshwater Research 34, 135-145. Miura, T., Yamamauchi, K., Takahashi, H., & Nagahama, Y. (1991). Human Chorionic Gonadotropin induces all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Developmental Biology 146, 258-262. Nilsson, L., Nyman, L., Westin, H. & Ornhagen, H. (1981). Simulation of the reproductive migration of European eels (Anguilla anguilla (L.)) through manipulation of some environmental factors under hydrostatic compression. Speculations in Science and Technology 4, 475-484. Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., & Hirose, K. (1996). Changes in fertilization and hatching rates with time after ovulation induced by 17α, 20β-dihydroxy-4-pregnen-3-one in the Japanese eel, Anguilla japonica. Aquaculture 139, 291-301. Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N. & Hirose, K. (1997). Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica. Fish Physiology and Biochemistry 17, 163-169. Palstra, A.P., van Ginneken, V., van den Thillart, G., 2004a. Artificial reproduction of the European silver eel (Anguilla anguilla L.). In: Aquaculture Europe 2004: Biotechnologies for Quality, pp. 641-642. EAS Special Publication No 34. Palstra, A.P., Cohen, E.G.H., Niemantsverdriet, P.R.W., van Ginneken, V.J.T., van den Thillart, G.E.E.J.M., 2004b Artificial maturation and reproduction of European silver eel: Development of oocytes during final maturation. Aquaculture, submitted. Pedersen, B.H., Ueberschär & Kurokawa, T.(2003). Digestive response and rates of growth in pre-leptocephalus larvae of the Japanese eel, Anguilla japonica. Fish Physiol.Biochem. 17, 163- 169.

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Pedersen, B.H., 2003. Induced sexual maturation of the European eel Anguilla anguilla and fertilisation of the eggs. Aquaculture 224, 323-338. Pedersen, B.H., 2004. Fertilisation of eggs, rate of embryonic development and hatching following induced maturation of the European eel Anguilla anguilla. Aquaculture 237, 461-473. Prokhorchik, G.A. (1986). Postembryonic development of European eel, Anguilla anguilla, under experimental conditions. J.Ichthyol. 26, 121-127. Prokhorchik, G.A., Petukhov, V.B. & Petrikov, A.M. (1987). Embryonic development of European eel, Anguilla anguilla, under experimental conditions. J.Ichthyol. 27, 37-43. Roberts, C.M. & Ormond, R.F.G. (1992). Butterfly social behaviour, with special reference to the incidence of territoriality: a review. Environmental Biology of Fishes 34, 79-93. Sebert, P. & Barthelemy, L. (1985). Effect of high hydrostatic pressure per se, 101 atm on eel metabolism. Respiration Physiology 62, 349-357. Simon, B., Sebert, P. & Barthelemy, L.(1988). Effects of long-term hydrostatic pressure per se (101 ATA) on eel metabolism. Can.J.Physiol.Pharmacol. 67, 1247-1251. Sugimoto, Y., Takeuchi, Y., Yamauchi, K. & Takkahashi, H. (1976). Induced maturation of female Japanese eels (Anguilla japonica) by administration of salmon pituitaries, with notes on changes of oil droplets in eggs of matured eels. Bull.fac.Fish.Hokaido University 27, 107-120. Wallace, R.A., Selman, K., 1981. Cellular and dynamic aspects of oocyte growth in teleosts. Amer. Zool. 21, 325-343. Yamamoto, K. & Yamauchi, K. (1974). Sexual maturation of Japanese eel and production of eel larvae in the aquarium. Nature 251, 220-221. Yamauchi, K., Nakamura, M., Takahashi, H. & Takano, K. (1976). Cultivation of larvae of Ja- panese eel. Nature 263, 412.

298 Annex 3

Publicity disseminations

(P) (professional) journals, (N) news papers, (R) radio & (T) TV.

P OVB-bericht (Organisatie ter verbetering van de Binnenvisserij) (1997) no.4:123-126. Een oceaanreis in de kelder. Door B.J.Lucas. N Leids Dagblad, 31 oktober 1997, pag 13. Marathon palingen in tunnel. Door Arno van 't Hoog. P Bionieuws, 18 november 1997 pag. 6. Zwemtunnel stuurt paling op virtuele paringstrek. Door Arno van 't Hoog. N Mare, Leids Universitair Weekblad pag. 1 en 2, 20 november 1997. Zesduizend kilometer voor de boeg in Leidse tunnels. Door Astrid Smit. N Reformatorisch Dagblad, 25 november 1997 pag. 17. Leidse onderzoekers gaan na of Europese paling de Sargassozee kan halen: Lange-afstandzwemmen in een kelder. Door drs.J.A.Coster. R Radiopraatje, Hilversum 2, AVRO, 26 november 1997. Interview met Marco Hopstaken. N De Limburger 9 december 1997 pag 19. Alarm: wie weet waar de paling paait ? Door Ron Buitenhuis. N Telegraaf 13 december 1997 pag 17. Zwemtocht van 6000 km in aquarium. Door Kees Roos. T TV-uitzending NOS-middageditie, 15 december 1997. Interview van Marcel Ouddeken met Dr.G.E.E.J.M.van den Thillart. N Eindhovens Dagblad 6 januari 1998 pag. 21. Wetenschap Alarm: Waar paait de paling ? Door Ron Buitenhuis. N NRC 9 januari 1998. De paringsdrift van de paling. Door Bram Pols. P Het Visblad NVVS, maart 98 pag 4& 5. Paling houdt onderzoekers bezig. T RTL4-journaal 19.30 h, dinsdag 26 mei 1998. Met de paling gaat het slecht maar Leidse Biologen proberen de paling voort te planten door de reis naar de Sargassozee in zwemtunnels te imiteren. N Mare, Leids Universitair Weekblad 35, 11 juni 1998. Leidse palingen redden het niet. Door Astrid Smit. N Leids Dagblad 16 juni 1998. Leidse Bioloog blijft optimistisch, virus gooit roet in eten.. Door Aad Rietveld. N Apeldoornse Courant, Arnhemse Courant, Deventer Dagblad, Gelders Dagblad, Overijssels Dagblad, pag. 5, 17 juni 1998, Paling zwemt 6000 km in Leids Laboratori- um. Door Menno Pols N Leeuwarder Courant. Paling zwemt 6000 km in een laboratorium, 20 juni 1998, zaterdagbijlage pag.9, door Menno Pols. N Provinciaalse Zeeuwse Courant. Leidse onderzoeker van Ginneken speurt naar mysterieuze palingtrek, Aal zwemt 6000 km in lab, varia pag.4, 17 juni 1998. Door Menno Pols. N Rotterdams Dagblad, Het grote mysterie van de palingtrek, kwekerijen hechten groot belang aan wetenschappelijk onderzoek, pag.4, 17 juni 1998. Door Menno Pols. N Amersfoortse Courant, Utrechts Dagblad. Voortplanting van paling is nog onbekend, Focus weten, pag. 17, 20 juni 1998, door Menno Pols. R radio interview Radio West, 22 juni 1998, interview Vincent van Ginneken met Linda Vosjan, Radio West over de oorzaken waarom de eerste proef met palingen in de zwemtunnels niet helemaal gelukt is.

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R radio interview Radio West, 29 juni 1998, interview Vincent van Ginneken met Linda Vosjan, Radio West over de hypothese dat PCB's mogelijk interfereren met de palingtrek en over de PCB-proef die van maart tot eind mei in zwemtunnels is uitgevoerd. P OVB-Bericht (organisatie ter verbetering van de Binnenvisserij). 1999 nummer 2: 50-51. Schieralen met electronica op OVB-proefbedrijf. Door Berry Lucas N Volkskrant, Wetenschap en Samenleving, 15 januari 2000 pagina 3w. Vette paling haalt Sargassozee best. T Noorderlicht, VPRO-televisie, dinsdag 8 februari 2000. uitzending over onder andere het Leidse palingonderzoek. P VPRO-gids, dinsdag 8 februari, pagina 59. Zonder fysieke inspanning geen voortplanting. P Mare, Leids Universiteitsblad, donderdag 20 januari pagina 7. Zesduizend kilometer zwemmen voor het nageslacht. Paargrage palingen. Door Bruno van Wayenburg. R Radiouitzending Faros, radio 5, Teleac, interview met Rob Buyter R Bespreking in nieuwsoverzicht VARA ‘Vroege Vogels’, Radio 16 januari. P Bionieuws, 29 januari 2000, nummer 2, pagina 1. Sanering vissersvloot onvermijdelijk, de paling ontglipt Nederland, Arno van het Hoog. N Intermediair 3, 20 januari 2000, pagina 137, De proefopstelling, door Astrid Smit. N Intermediair 6, 10 februari 2000, pagina 129-131, Wetenschap en Techniek. De dramatische val van de paling, door Astrid Smit. T TV special on eel, 25 min. (jan 2001) Het geheim van de paling. T TV interview (Sept 2002) Program ‘Iel’ of Omrop Fryslân P Bionieuws 20, 28-11-2003: Paling plant zich voort in Leids laboratorium P Bionieuws 20, 28-11-2003: Zesduizend kilometer op een pakje boter N Mare 04/12/03: Leidse aal laat zich kweken R Radio West 05/12/03 (interview) N 4 different newspapers (IJmuider Courant, Leidsch Dagblad, Gooi & Eemlander, Haarlems Dagblad): 06/12/03: Doorbraak met kweek van paling N Haagsche Courant 06/12/03: Leidse zoölogen melden doorbraak palingkweek.

N Noordhollands Dagblad 08/12/03: Doorbraakje in duistere wereld van de paling N IJmuider Courant, Leidsch Dagblad, Gooi & Eemlander, Haarlems Dagblad P Leidraad 01/04: Leidse aal laat zich kweken N Telegraaf 05/04 Nederlandse Biologen kweken palingembryo's N Goudse Courant (05/2004) Paling in Leids lab gekweekt T TV NOVA 05/04 and 08/04 Broodje paling gered (20 min special on eel) T German TV (VOX) 30-min broadcast of eel film made by Tesche (december 2004) T Omroep Zeeland (28/1/2005) EVEX virus N Algemeen Dagblad (5/2/2005) Virus oorzaak van slechte palingstand N Trouw (6 /2/2005) Palingen bezwijken aan virus R KRO radio, interview (7/2/2005) Vroege vogels: Palingvirussen N Le Monde (June 18, 2000) . L'anguille, une énigme argentée enfouie en eaux profondes. By Catherine Vincent. N Article in the daily newspaper 'Het Belang van Limburg'. p. 4. (12 october 2004.). “Binnen vijf jaar is er geen paling meer”. T Participation in a “Tesche documentary productions” film on the eel (10/04) N Publicity (half a page) in a daily newspaper to inform about the Brest EELREP meeting (07/04)

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List of publications

1. Van Ginneken, V.J.T.; Nouws, J.F.M.; Grondel, J.L.; Driessens, F.; and Degen, M. (1991). Pharmacokinetics of sulphadimidine in carp (Cyprinus carpio L.) and rainbow trout (Salmo gairdneri Richardson) acclimated at two different temperatures. The Veterinary Quarterly 13:88-96. 2. Komen, J.; Wiegertjes, G.F.; van Ginneken, V.J.T.; Eding, E.H.; Richter, C.J.J. (1992). Gynogenesis in common carp (Cyprinus carpio, L.) 111. The effects of inbreeding on gonadal development of heterozygous and homozygous gynogenetic offspring. Aquaculture 104:51-66. 3. Nouws, J.F.M.; van Ginneken, V.J.T.; Grondel, J.L.; Degen, M. (1993). Pharmacokinetics of sulphadizone and trimethoprim in carp (Cyprinus carpio L.) acclimated at two different temperatures. J.Vet.Pharmacol.Therap. 16:110-113. 4. Van Ginneken, V.J.T.; Gluvers, A.; van der Linden, R.W.; Addink, A.D.F.; van den Thillart, G.E.E.J.M. (1994). Direct calorimetry of aquatic animals: automated and computerised data-acquisition system for simultaneous direct and indirect calorimetry. Thermochimica Acta 247:209-224. 5. Van Ginneken, V.J.T.; Vanderschoot, J.; Addink, A.D.F.; van den Thillart, G.E.E.J.M. (1995). Direct calorimetry of aquatic animals: dynamic response of biological processes. Thermochimica Acta 249:143-159. 6. Van Ginneken, V.; van den Thillart, G.; Addink, A.; Erkelens, C. (1995). Fish muscle energy metabolism measured during hypoxia and recovery: an in vivo 31P-NMR study. Am.J.Physiol.268: R1178-R1187. 7. Van Ginneken, V.; Nieveen, M.; van den Thillart, G.; Addink, A. (1995). Neurotransmit- ter levels and energy status in brain of fish species with and without the survival strategy of metabolic depression: evidence for a role of GABA in metabolic depression. Comp.Bio- chem.Physiol 114A : 189-196. 8. Van Ginneken, V.; Addink, A.; van den Thillart, G. (1996). Direct calorimetry of aquatic animals. Effect of acidification on heat production and oxygen consumption of Tilapia (Oreo- chromis mossambicus Peters). Thermochimica Acta 276:7-15. 9. Van Ginneken, V.; van den Thillart, G.; Addink, A.; Erkelens, C. (1997). Synergistic effect of acidification and hypoxia: an in vivo 31P-NMR study and respirometric study. Am.J. - Physiol. 271, R1746-R1752. 10. Van Ginneken, V.; Eersel van, R.; Balm, P.; van den Thillart, G. (1997). Tilapia is able to withstand long-term exposure to low environmental pH, judged by their energy status, ionic balance and plasma cortisol. J.Fish Biology 51: 795-806. 11. Van Ginneken, V.; Addink, A.; van den Thillart, G.; Körner, F.; Noldus, L.; Buma, M. (1997). Metabolic rate and level of activity determined in tilapia (Oreochromis mossambicus Peters) by direct and indirect calorimetry and video monitoring. Thermochimica Acta 191:1-13. 12. Van Ginneken, V.; van Cauwbergh, P. Nieveen, M., van den Thillart, G.; Addink, A. (1998). Influence of hypoxia exposure on the energy metabolism of common carp. (Cyprinus carpio L.). Neth.J.Zool. 48:65-82. 13. Van Ginneken, V.; G.van den Thillart, Muller, H.; S.van Deursen, M.Onderwater, J.Visee, V.Hopmans, G.van Vliet, Nicolay, K. (1999). Phosphorylation state of red and white muscle in tilapia during graded hypoxia: an in vivo 31P-NMR study. Am.J.Physiol. 277: R1501- R1512. 14. Vincent van Ginneken and Guido van den Thillart (2000). Eel fat stores are enough to reach the Sargasso. Nature 403:156-157.

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15. Ed van der Meijden, Catharina A.M. van der Veen-van Wijk, Vincent van Ginneken (2000). Cotesia (Apanteles) Popularis L. parasitoids do not always kill their host. Entomologist's monthly magazine 136:117-120. 16.Vincent J.T. van Ginneken, Marjolijn Onderwater, Olga Lamua Olivar, and Guido E.E.J.M. van den Thillart (2001). Metabolic depression and investigations of glucose/ethanol conversion in the European eel (Anguilla anguilla Linnaeus 1758) during anaerobiosis. Thermochimica Acta 373: 23-30. 17. V.van Ginneken, P.Balm, V.Sommandas, M.Onderwater, G.van den Thillart (2002). Acute stress syndrome of the yellow European eel (Anguilla anguilla Linnaeus) when exposed to a graded swimming-load. Neth.J.Zoology 52: 29-42. 18. Ozorio, R.O.A.; Verstegen, M.W.A.; van den Briel, V.; van Ginneken, V.J.T.; Verreth, J.A.J., Huisman, E.A. Dietary carnitine restores white muscle energy reserves of exercised African catfish (Clarias gariepinus) juveniles. Chapter 6 In: Dietary L-carnitine and energy and lipid metabolism in African catfish (Clarias gariepinus) juveniles. PhD-thesis, Agricultural University Wageningen, November 2001, ISBN 90-5808-493-0. 19. Van den Thillart, van Ginneken, V.; Körner, F.; Heijmans, R.; van der Linden, R.; Gluvers (2004). Endurance swimming of European eel. J.Fish.Biology 65: 312-318. 20. Van Ginneken, Snelderwaard, P.; van der Linden, R.; van der Reijden, N.; van den Thillart G.; Kramer, K. (2004). Coupling of heart rate with metabolic depression in fish: a radio-telemetric and calorimetric study, Thermochimica Acta 414: 1-10.. 21. Van Ginneken, V.; Boot, R.; Murk, T., van den Thillart, G.; Balm, P. (2004). Blood- plasma substrates and muscle lactic-acid response of the common carp and trout: indications for a limited lactate-shuttle. Animal Biology 54: 119-130. 22. Van Ginneken, V.; Haenen, O.; Coldenhoff, K.; Willemze, R.; Antonissen, E.; van Tulden, P.; Dijkstra, S.; Wagenaar, F.; van den Thillart, G. (2004). Presence of eel viruses in eel species from various geographic regions. Bull.Eur.Ass.Fish.Path. 24(5):270- 274. 23. Palstra, A.P., van Ginneken, V., van den Thillart, G., (2004). Artificial reproduction of the European silver eel (Anguilla anguilla L.). EAS Special Publication No 34. 24. Van Ginneken, V.; Muusze, B.; Klein Breteler, J.; Jansma, D.; van den Thillart, G. (2005). Microelectronic detection of activity level and magnetic orientation of yellow European eel (Anguilla anguilla L.) on a pond, Environmental Biology of Fishes, 72(3):313-320.. 25. V.van Ginneken, E.Antonissen, U.K.Müller, R.Booms, E.Eding, J.Verreth, G.van den Thillart (2005), Eel migration to the Sargasso: remarkably high swimming efficiency and low energy costs. J.Exp.Biol., 208: 1329-1335. 26. Van Ginneken, B.Ballieux, V.; Willemze, R.; Coldenhoff, K.; Lentjes, E.; Antonissen, E., Haenen, O.; G.van den Thillart (2005). Hematology patterns of migrating eels and the role of EVEX virus. Comp.Biochem.Physiol. C, 140: 97-102. 27. A.Palstra, S.Szekely, V.van Ginneken, G.van den Thillart (2005). Swim fitness of European eel (Anguilla anguilla). Comparative Biochemistry and Physiology Part A, 141, S163-S173 28. A.P.Palstra, E.G.H.Cohen, P.R.W.Niemantsverdriet, V.J.T.van Ginneken, G.E.E.J.M. van den Thillart (2005) Artificial maturation and reproduction of European silver eel: development of oocytes during final maturation. Aquaculture 249: 533-547. 29. V.van Ginneken, G.Vianen, B.Muusze, L.Verschoor, O.Lugten. M.Onderwater, S.van Schie, P.Niemantsverdriet, R.van Heeswijk, E.Eding, G.van den Thillart (2005). Gonad development and spawning behavior of artificially matured European eel (Anguilla anguilla L.). Animal Biology, 55: 203-218. 30. Palstra, A.P., van Ginneken, V.J.T., Murk, A.J., van den Thillart, G.E.E.J.M. (2006) Are dioxin-like contaminants responsible for the eel (Anguilla anguilla) drama? Naturwissenschaften, 93:145-148.

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31. R. Ozório; V. van Ginneken, G.van den Thillart, M.Verstegen, J.Verreth. (2005). Dietary carnitine maintains energy reserves and delays fatigue of exercised African catfish (Clarias gariepinus) fed high fat diets. Sci.Agric. 62(3): in press. 32. V.van Ginneken. and G. Maes, The European eel (Anguilla anguilla, Linnaeus), its lifecycle, evoltion and reproduction: a literature review. Reviews in Fish and Fisheries, in press. 33. P.Ch. Snelderwaard, V. van Ginneken, F. Witte, H.-P. Voss, and K. Kramer. Surgical procedures for implanting a radio-telemetry transmitter to monitor ECG, heart rate and body temperature in small Goldfishes and Gold carps under laboratory conditions, Lab. Animal, accepted. 34. V.van Ginneken, B.Ballieux and G. van den Thillart. Direct calorimetry of free moving eels with manipulated thyroid status. Naturwissenschaften, accepted. 35. V.van Ginneken, S.Dufour, M.Shaihi, P.Balm, K.Noorlander, M.de Bakker, J.Doornbos, E.Antonissen, I.Mayer, G.van den Thillart. A 5,500-km swim trial stimulates gonad maturation in the European eel (Anguilla anguilla L.). Gen.Comp.Endocrinol., resubmitted after revision. 36. V.van Ginneken; C.Durif; P.Balm; K.M. Verstegen, E R.Boot; E. Antonissen, G.van den Thillart: Silvering of European eel (Anguilla anguilla L.): seasonal changes of morphological and metabolic parameters Animal Biology., submitted.. 37. V.van Ginneken; C.Durif; S.Dufour; M.Sbaihi; R.Boot; K.Noorlander J.Doornbos, A.J..Murk. G.van den Thillart: Endocrine and metabolic profiles during silvering of the European eel (Anguilla anguilla L.): Animal Biology, submitted. 38. V.van Ginneken, K.Coldenhoff, J.Hollander, F.Lefeber and G. van den Thillart. Depletion of energy stores in white muscle and not internal acidosis may be a cause for fish mortality after strenuous exercise: an in vivo 31P NMR-study. J.Exp.Biol., submitted. 39. V.van Ginneken, Kiihne, S.; van Dijk, K.W.; Ham, L.; Lefeber, F.; Erkelens, K.; Duivenvoorden, I.; Voshol, P.; Havekes, L.; Poelmann, R. .(2006). Liver fattening during famine and feast, an evolutionary paradox. A localized MRI-spectroscopy study. Proceedings, ISMRM 14th Scientific Meeting Seattle, Washington, USA

Manuscripts in preparation

40. V.van Ginneken, Palstra, A.; Leonards, P.; Nieveen, M.; van den Berg, H.; Flik, G.; Spanings, T.; Niemantsverdriet, P.; Murk, A.; van den Thillart, G. Effects of PCBs on the energy cost of migration and blood parameters of European silver eel (Anguilla anguilla L.). To be submitted to The Journal of Experimental Biology. 41. V.van Ginneken, Kiihne, S.; van Dijk, K.W.; Ham, L.; Lefeber, F.; Erkelens, K.; Duivenvoorden, I.; Voshol, P.; Havekes, L.; Poelmann, R. .(2006). The dynamics of Hepatic steatosis is response to fasting and fatty diet measured in vivo in mice with localized 1H magnetic resonance spectroscopy (1H MRS): Validation of the method. To be submitted to J.Biol.Chem. 42. V.van Ginneken, Ham, L.; van Dijk, K.W.; van der Greef, J.; Verheij, E.; Ramakers, R.; Voshol, P.; Havekes, L.; van Eck, M.; Poelmann, R. Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study at hepatic steatosis. To be submitted to Journal of Cellular Physiology. 43. Palstra, A.P., van Ginneken, V.J.T., van den Thillart, G.E.E.J.M. Swim fitness of European silver eels (Anguilla anguilla). To be submitted to The Journal of Experimental Biology.

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44. Palstra, A.P., Heppener, D.F.M., van Ginneken, V.J.T., Székely, C.,van den Thillart, G.E.E.J.M. Swim efficiency and reproductive migration of silver eels are severely impaired by the swim-bladder parasite Anguillicola crassus. To be submitted to The Journal of Experimental Biology. 45. Palstra, A.P., Curiel, D., Fekkes, M., de Bakker, M., van Ginneken, V.J.T., van den Thillart, G.E.E.J.M. Swimming stimulates silvering and oocyte development of European eel (Anguilla anguilla). To be submitted to The Canadian Journal of Aquatic Sciences and Fisheries. 46. Palstra, A.P., Antonissen, E., Clavero, M.E., Nieveen, M., Niemantsverdriet, P., van Ginneken, V.J.T., van den Thillart, G.E.E.J.M. The fate of fat in silver eels: lipid requirements for migration and maturation. To be submitted to The Journal of Fish Biology.

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Curriculum Vitae

Vincentius Johannes Theodor van Ginneken was born in December 23, 1963, Amsterdam, The Netherlands. Primary school was performed in this city. Secondary education at the Grammar school; was performed during the period 1976-1977, at the Fons Vitae College, Amsterdam and during the period 1977-1982 at the Van Maerlantlyceum, Eindhoven, The Netherlands. A University study was followed during the period 1982-1989, at the Agricultural University, Wageningen, field of education: “Animal Husbandry”, specialisation Aquaculture.

His graduation topics for his MSc were: 1. 'Fish diseases' NUFFIC/UNIBRAW/LUW/Fish-project, Malang, Indonesia. Supervisors: Dr. J.H. Boon, Ir. N. Zonneveld, and Ing. W.J.A.R. Viveen. 2. 'The pharmacokinetic behaviour of antibiotics in fish' Supervisors: Prof. Dr. W.B. van Muiswinkel, Dr. J.L. Grondel, and Dr. J.F.M. Nouws. 3. 'The development of inbred carp lines using gynogenesis' Supervisors: Prof. Dr. E.E.A. Huisman, Dr. C.J.J. Richter, and Dr.Ir. J. Komen.

During the period 1990-1995 he performed a first PhD-research topic at the Faculty of Mathematics and Natural Sciences, Leiden University, The Netherlands. The research group was the department of Animal Physiology, Biology, Institute of Evolutionary and Ecological Sciences (EEW). The project was subsidised by the Netherlands Organisation for Scientific Research (NWO). This resulted in a Ph-D-dissertation: The effects of adverse environmental factors like hypoxia and acidification on the energy metabolism of fish: an in vivo 31P-NMR and calorimetric study. 336 pp. Promotor: Prof.Dr. A.D.F. Addink, Copromotor: Dr. G.E.E.J.M. van den Thillart.

Based on two postdoc fellowships (STW-grant LBI66.4199 and an EU-grant QLRT-2000- 01836, see below) a second PhD-dissertation was performed at Biology, Leiden University “Simulated migration of European eel” (Anguilla anguilla, Linnaeus 1758). Promotor: Prof.Dr. J.Verreth, Fish culture and Fisheries, Wageningen Agricultural University, The Netherlands; Copromotor: Dr. G.E.E.J.M. van den Thillart, Biology, Leiden University, The Netherlands. The defence of this thesis will be at the Faculty of Agricultural Sciences, Wageningen Agricultural University, 14 June 2006, 13.30 h.

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Scientific work experience: -1986-1988: part-time assistantship for the Netherlands University Foundation For International Corporation for the NUFFIC/UNIBRAW/LUW/Fish-project, Malang, Indonesia, Agricultural University Wageningen, supervisor Dr. Carel Richter. -1990-1995: PhD-thesis period, Biology, Leiden University, supervisor prof.Dr. Ab Addink. -1996: Research-project "heat shock proteins" Animal Physiology, Biology, Leiden University, supervisor Dr. Guido van den Thillart. -1997-2000: Postdoc, STW-project No. LBI66.4199: Simulated migration of European eel. Effect of long term swimming and parasitic infection pressure on energy balance and gonadal development, Biology, Leiden University, supervisor Dr. Guido van den Thillart. -2001: Postdoc, PCB experiment eels sponsor EUROCHLOR, supervisor Dr.Tinka Murk, and reproduction of eel, sponsor: Organisation for Improvement of Inland Fisheries (OVB), supervisor Dr. Lex Raat, Research was performed Biology, Leiden University. -2002-2003, postdoc on an EC-project, 'Quality of Life and management of Living Resources', "Estimation of the reproduction capacity of European eel" QLRT-2000-01836, Leiden University, supervisor Dr. Guido van den Thillart..

-2003- 2005, postdoc, ‘Centre for Medical Systems Biology’ (CMSB), supervisor prof.Dr.Rob Poelmann, Magnetic Resonance Microscopy (MRI) with transgenic mice models (disease models: Migraine, Depression, Alzheimer, Metabolic Syndrome), Leiden University Medical Centre (LUMC). During this postdoc period I get interested in human diseases like Alzheimer, metabolic syndrome, migraine and depression and its common denominators often related to life style and nutrition. My research interest was mainly focussed the last 2 years on obesity (metabolic syndrome) resulting in diabetes 2, hepatic steatosis due to a fatty diet (measured with localized MRI spectroscopy techniques). And the search for biomarkers in obesity with mass spectrometry. Also additionally postdoc MRI work was performed for TIBOTEC/TNO at the effects of anti-HIV cocktails on the fat distribution in a mice model.

2006-present: writing down the presented thesis: “Simulated Migration of European Eel”

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Dankwoord

5500 km zwemmen als activiteit was toch wel een moeizame aangelegenheid waarbij ik geplaagd werd door discontinuϊteit in aanstelling door de 40% bezuiniging op het personeel bij Biologie Leiden. Er zijn tijdens de zwemreis momenten geweest dat ik met de ‘Wetenschap’ wilde stoppen, maar 2 keer een tophypotheek op mijn koophuis, en een half jaar als Dr. Ir. op Schiphol vliegtuigen in- en uitladen tussen mensen met alleen een vorkheftruckdiploma gaven me weer wat adem en financiёle armslag om door te gaan. 2 % van de gepromoveerden krijgt maar een vaste aanstelling aan een Nederlandse Universiteit (Bionieuws Mei 2006). Ik moet hier even aarzelen of ik een Leids ‘snikken of grimlachjes’ op mijn gezicht zal toveren. Deze tweede promotie is dan ook niet onstaan uit ijdelheid maar puur als een poging om in ‘de race’ te blijven en de kansen op een vaste baan te vergroten.

Goed dit is een dankwoord, laat ik beginnen met de anonieme referenten te bedanken die mij een paar jaar geleden het STW-project gegund hebben, de basis voor het aalonderzoek. Zonder de financiele bijdrage van Ir.Jan van Rijsingen (Royaal BV, Helmond) als hoofdsponsor in de gebruikerscommisie was dit project nooit gehonoreerd. Mijn promotor Prof.Dr. Johan Verreth, beste Johan, bedankt dat je mij de gelegenheid gaf om bij je te promoveren en dat je deze ceremonie georganiseerd hebt. Mijn copromotor beste Guido. Ik dank je voor je kritische wetenschappelijke houding, voor m’n aanstelling binnen EELREP waardoor ik dit proefschrift heb kunnen afmaken en voor alles wat ik van je heb geleerd tijdens beide promoties. De hoge eisen die je stelt aan jezelf maar ook aan je medewerkers hebben uiteindelijk tot onder andere dit product geleid. Mijn (ex)collega’s van Integratieve Zoologie: Christoph Bagowksi, Kees Barel, Howard Berger, Merijn de Bakker, Pieter Gaemers, Maarten-Jan van Hasselt, Richard van Heeswijk, Daniёl Jansma, John Jeffrey, Mourik Mietes, Bie Muusze, Patrick Niemantsverdriet, Maaike Nieveen, Kees Noorlander, Marjolijn Onderwater, Arjan Palstra, Michael Richardson, Arjan Rozier, Carlo Rutjes, Sjoerd van Schie, Peter Snelderwaard, Guido van den Thillart, Gerjanne Vianen-de Mooij, Ardi Visser, Frans Witte en Francis Zitman. Dat jullie me met al mijn hebbelijkheden en onhebbelijkheden hebben geaccepteerd. Die speciale filmnacht in het Kamerlingh Omnes gebouw waarbij ik maniakaal was na twee nachten bijna niet geslapen te hebben omdat de voorbereidingen om afpaaiende palingen te filmen zoveel tijd hadden gekost zou een prachtige komedie geweest zijn. Eigenlijk had jij Bie toen de de filmcamera op mij moeten richten en niet op de palingen. Ik schaam me er nog steeds een beetje voor. Herman Berkhout en Jos Onderwater, nog bedankt voor de prachtige foto’s van de afpaaiende palingen en jij Bie voor de film. Ik zal niet al mijn collega’s noemen maar voor enkelen een speciaal woord. -Prof.Dr. Mike Richardson, bedankt voor het corrigeren van het Engels van de manuscripten en dat je mij de gelegenheid gaf het proefschrift binnen je groep af te maken. -Arjan Palstra, beste Arjan, de congressen waren gezellig. Je bent een goed en creatief onderzoeker dus het palingwerk is in veilige handen bij je. -Peter Snelderwaard, beste Peter, die nacht dat bij een zwemproef die al enkele maanden liep de stroom uitviel en we de hele nacht met de Technische Dienst met onze enkels in het water stonden om de tunnels weer op gang te krijgen zal ik nooit vergeten. Jij stond midden in de nacht klaar om te helpen. Mijn dank. -Maaike Nieveen, lieve Maaike: we zijn al vanaf het begin in de Dierfysiologie en later IZ groep met al z’n ups en downs. We kennen elkaar nu wel zo’n beetje zonder al teveel woorden.

307 Dankwoord

-Francis Zitman, lieve Francis, bedankt dat je me altijd bij het IZ gebeuren bent blijven betrekken. Ook toen het werk voor mij op het LUMC was beёindigd. Patrick Niemantsverdriet, beste Patrick en Ira: Jullie vervulden een centrale rol in de gezellige dingen die we deden met de studenten zoals het voetbal op dinsdag en het naborrelen op vrijdag. Ik hoop dat we die tradities nog lang zo zullen houden. -Daniel Jansma, beste Daan: Bedankt voor alle werk wat je in de loop der jaren hebt gedaan. Ook bij de afronding van het promotiewerk. De laatste 1000 km zwemmen viel me zwaar. Bedankt voor al het lay-out werk. Ik ben blij dat jij op het LUMC nu je stek gevonden heb en Chamindi kan laten overkomen. Mijn studenten. Het zijn er vele geweest. Ik kan niet alle werk opnoemen dat jullie verricht hebben. Daar moeten de mensen maar voor op de co-auteurschappen kijken van de diverse (pre)-publicaties. Ik noem, Vinod Sommandas, Ron Boot, Corine Houtman, Karen Coldenhoff, Erik Antonissen, Louise Verschoor, Olivier Lugten en Chris Sakalis. Ik wil speciaal Erik Antonissen bedanken voor de ongelooflijke hoeveelheid werk die je verricht hebt in getallenverwerking en statistiek. De studenten van de laboratoriumschool voor het histologisch werk: Jorg Doornbos, Sjoerd van Schie en Frits Moulijn. Ook de diverse coauteurs op de artikelen wil ik danken voor hun bijdrage. De mensen van de technische dienst (fijnmechanica en electronica) Frans Körner, Rob van der Linden, Ab Gluvers, Rinus Heijmans, Jeroen Mesman, Frits van Tol en Gerard Kostense die de tunnels hebben gebouwd en het meetregistratiesysteem op de vijver in Beesd. Winston Churchill zei eens (in een heel andere context tegen de Amerikanen), “Give us the tools and we will finish the job”. Jullie hebben me de meetinstrumenten gegeven. De mensen van de computerdienst, Hugo Nijhof, Koos Weerlee. Mensen van de tekenafdeling, uit de Gorlaeus tijd, Ton Huigen en op het van der Klaauw, Martin Brittijn. Dank voor de ongelofelijke hoeveelheid werk die jullie hebben verricht met de tekeningen voor de publicaties. Het voetbalelftal van Biologen op dinsdagavond en de kroegtijgers op vrijdag. Patrick, Ira, Marieke, Hanneke, Irvil, Arjan, Daniel, Maarten, Machteld, Remko, Danielle, Leon, Olga, Merijn, Monique, Ilse, Chris, Angela en (ex)studenten zoals Marjolijn, Barbara, Chris en anderen. Dinsdag was gezond, vrijdag was ongezond om lekker brak het weekend in te stappen. Mijn familie, de reden waarom ik nog niet naar het buitenland ben gegaan. Mijn allerliefste vader, dank dat je (ondanks de ernstige problemen met je eigen gezondheid) altijd voor me klaar stond. Mijn lieve tante To, bedankt dat je altijd een oogje in het zeil heb gehouden hoe het met Pap ging. Mijn zus Mariette en zwager Edward. De traditie die jullie in ere hielden om net als Mamma deed om op verjaardagen Indische rijsttafels te blijven koken heb ik zeer gewaardeerd. Tot jullie kinderen Simon, Veertje en Jelle wil ik zeggen ga nooit Biologie studeren, daar is geen droog brood in te verdienen. Mijn broer Stephan, schoonzus Marianne en Famke. Dank voor de gastvrijheid bezorgdheid en medeleven. Tot Stef een speciaal woord, lieve buddy, lieve broer. Je opbeurende woorden zijn altijd heel belangrijk voor me geweest. Marieke van Schie en Hans Rohlof voor de adviezen en ondersteuning. Lieve Louise en Kaitje, bedankt dat jullie me in bepaalde mate toelieten in jullie leven. Gezamelijk met elkaar eten en leuke dingen doen waren voor mij lichtpuntjes in de week. Beste Ramon en Qurra, ik getuige op jouw huwelijk, jij nu paranimf. We kennen elkaar al heel lang. Je bent nog student bij me geweest. De fitnesstrainingen op donderdag voorafgegaan door een goed etentje waren prima om de endorfines weer wat door het lijf te laten jagen. Lieve Tamara, ook jou ken ik nog uit de tijd van Ramon. Leuk dat we altijd contact zijn blijven houden. Ik heb je optimisme altijd bewondert en als voorbeeld gesteld.

308 Dankwoord

Hanneke, de danspartijtjes zondagochtend in de ‘Chill-out’ waren onvergetelijk. Ik voelde me altijd heel schuldig als we naar huis gingen en we mensen naar de kerk zagen gaan. Beste Wouter (en Annemiek) en Erik, oud-afdelingsgenootjes uit mijn Wageningse tijd. We hebben vroeger veel gepraat in onze studententijd over het boek “Bij nader inzien” van Voskuil. Het echte leven is niet alleen maar dromen maar ook concessies doen. Vorige keer was jij paranimf Wouter, nu jij Erik. Beste Niels (en Marion), Richard (en Annemarie) en Bart, oud-Wageningers. De tradities die we in stand hielden om elkaar een paar keer per jaar te zien met Carnaval, Leids Ontzet of Wageningen Bevrijdingsdag moeten we in ere houden. Oude vriendschappen blijven toch steunpunten in het leven. En nog verder terug uit de Middelbare Schooltijd, Hans Melis (en Sabine), ik waardeer het contact dat we zijn blijven houden. Enkele mensen van Anatomie (LUMC) waar ik 1.5 jaar gewerkt heb wil ik bedanken. Prof.Dr.Rob Poelmann, Prof.Dr.Adri Gittenberger, mijn kamergenoten Pauline Roest, Bianca Hogers, Angela Engel en 1 kamer erboven Paul Gobée.

Iedereen bedankt !!!!!

Verder wil ik de volgende sponsors hartelijk bedanken voor hun bijdrage in de drukkosten van dit proefschrift:

* Nutreco Aquaculture afdeling “Skretting Northwest Europe”

* Sportvisserij Nederland

* Gebroeders Kraan, Palingimporteur, Urk.

* Cooperative Producentenorganisatie Nederlandse Vissersbond IJsselmeer U.A.

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